Cell-specific involvement of HNF-1beta in alpha 1-antitrypsin gene expression in human respiratory epithelial cells

Chaobin Hu and David H. Perlmutter

Departments of Pediatrics and Cell Biology and Physiology, Washington University School of Medicine, and Division of Gastroenterology and Nutrition, St. Louis Children's Hospital, St. Louis, Missouri 63110


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The synergistic action of hepatocyte nuclear factor (HNF)-1alpha and HNF-4 plays an important role in expression of the alpha 1-antitrypsin (alpha 1-AT) gene in human hepatic and intestinal epithelial cells. Recent studies have indicated that the alpha 1-AT gene is also expressed in human pulmonary alveolar epithelial cells, a potentially important local site of the lung antiprotease defense. In this study, we examined the possibility that alpha 1-AT gene expression in a human pulmonary epithelial cell line H441 was also directed by the synergistic action of HNF-1alpha and HNF-4 and/or by the action of HNF-3, which has been shown to play a dominant role in gene expression in H441 cells. The results show that alpha 1-AT gene expression in H441 cells is predominantly driven by HNF-1beta , even though HNF-1beta has no effect on alpha 1-AT gene expression in human hepatic Hep G2 and human intestinal epithelial Caco-2 cell lines. Expression of alpha 1-AT and HNF-1beta was also demonstrated in primary cultures of human respiratory epithelial cells. HNF-4 has no effect on alpha 1-AT gene expression in H441 cells, even when it is cotransfected with HNF-1beta or HNF-1alpha . HNF-3 by itself has little effect on alpha 1-AT gene expression in H441, Hep G2, or Caco-2 cells but tends to have an upregulating effect when cotransfected with HNF-1 in Hep G2 and Caco-2 cells. These results indicate the unique involvement of HNF-1beta in alpha 1-AT gene expression in a cell line and primary cultures derived from human respiratory epithelium.

protease inhibitors; pneumocytes; tissue-specific gene expression


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha 1-antitrypsin (alpha 1-AT) is an ~55-kDa serum glycoprotein that inhibits the destructive neutrophil proteases elastase, cathepsin G, and proteinase 3 (28). It is the archetype of a family of serum proteins, many of which are serine protease inhibitors and are therefore called serpins (6, 18). Lack of elastase inhibitory activity in the lung is thought to be responsible for the predisposition of alpha 1-AT-deficient individuals to destructive lung disease/emphysema (20). Moreover, functional inactivation of alpha 1-AT by active oxygen intermediates released during cigarette smoking is believed to play a role in pulmonary emphysema in alpha 1-AT-sufficient individuals (20). A subgroup of individuals with the classical form of alpha 1-AT deficiency are predisposed to liver injury and hepatocellular carcinoma presumably because of the hepatotoxic effect of the mutant alpha 1-AT molecule within liver cells (28).

Plasma alpha 1-AT is predominantly derived from the liver, as shown by studies of changes in alpha 1-AT allotypes after orthotopic liver transplantation (1, 16). Synthesis of alpha 1-AT is abundant in human hepatoma cell lines and in human hepatocytes in primary culture, and alpha 1-AT mRNA is extremely abundant in hepatocytes in human liver, as determined by in situ hybridization analysis (26). There is also evidence for extrahepatic sites of synthesis, including blood monocytes, tissue macrophages, and intestinal epithelial cells (26, 29). Our recent studies have indicated that expression of the alpha 1-AT gene in hepatocytes and enterocytes is predominantly driven by the synergistic action of hepatocyte nuclear factor (HNF)-1alpha and HNF-4 (17). HNF-1alpha plays an important role in the increase in alpha 1-AT gene expression that accompanies differentiation of enterocytes from crypt to villous tip. HNF-1beta , in the absence or presence of HNF-4, has no effect on alpha 1-AT gene expression in hepatocytes or enterocytes (17).

Several recent studies have indicated that alpha 1-AT is also synthesized in human pulmonary airway epithelial cells, including the Calu-3 and A549 cell lines and normal bronchial epithelial cells in primary culture (9, 34). Previous studies have shown alpha 1-AT mRNA in bronchial and bronchiolar epithelial cells of human fetal lung by in situ hybridization analysis (22). Expression of alpha 1-AT by airway epithelial cells could potentially constitute an important component of the antiprotease defense system locally within the lung.

In this study, we used the human pulmonary epithelial cell line H441 as well as nuclear extracts and RNA from human respiratory epithelial cells in primary culture to examine the possibility that alpha 1-AT gene expression in pulmonary epithelial cells is directed by the synergistic action of HNF-1alpha and HNF-4 and/or by the action of more pneumocyte-specific transcription factors such as HNF-3, which have been shown to play an important role in gene expression in pneumocytes (3-5, 30).


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

Plasmid constructs. Progressive deletions of the human alpha 1-AT promoter region from -1951 to -2 were generated by PCR amplification using the human genomic alpha 1-AT clone hAAT7zf (kindly provided by Dr. K. Ponder, St. Louis, MO) as template. These PCR fragments were subcloned into the KpnI/HindIII site of the pGL3 basic luciferase reporter vector (Promega, Madison, WI) to generate alpha 1-AT promoter-luciferase fusion plasmids. The Mut-2 plasmid, which is an alpha 1-AT (-137 to -2) promoter-luciferase chimera with a mutation in the HNF-1-binding site, has been previously described (17). Our previous electrophoretic mobility shift assay (EMSA) studies showed a marked decrease in binding of HNF-1alpha and HNF-1beta to this plasmid compared with the plasmid without alterations in the HNF-1-binding region (17). The Mut-3 plasmid, which contains a mutation in HNF-3 binding, was generated for these studies. For this plasmid, we altered two nucleotides in the HNF-3-binding site at -101 to -93 (5'-TGTTTGCTC-3') within the alpha 1-AT (-137 to -2)-promoter luciferase chimera. The highly conserved thymidines at -101 and -97 were converted to adenines. Results of EMSA indicated that these mutations disrupted binding to nuclear proteins in extracts from Hep G2, Caco-2, and H441 cells (data not shown).

Murine HNF-1alpha and HNF-1beta expression plasmids (25) provided by Dr. T. C. Simon (St. Louis, MO) were subcloned into the pSG5 plasmid (Stratagene, La Jolla, CA). Full-length HNF-4 cDNA subcloned into the pMT2 expression vector (31) was provided by Dr. J. Rottman (St. Louis, MO). HNF-3alpha and HNF-3beta expression plasmids pPac HNF-3alpha and pPac HNF-3beta (12) were provided by Dr. G. Suske (Marburg, Germany). The pRL-TK vector was purchased from Promega.

Cell culture, DNA transfections, and luciferase assay. The Hep G2, HeLa, and Caco-2 cells were grown as previously described (26). H441 cells were purchased from American Type Culture Collection and maintained in RPMI 1640 medium with 2 mM L-glutamine, sodium bicarbonate (0.5 g/l), 10 mM HEPES, 1.0 mM sodium pyruvate, and 10% fetal bovine serum. This cell line is derived from a human pulmonary papillary adenocarcinoma. It expresses surfactant proteins A and B. Electron-microscopic analysis shows the presence of multilamellar bodies and cytoplasmic structures resembling Clara cell granules in these cells (27). The promoter-luciferase plasmids were cotransfected with the indicated amount of HNF-1alpha , HNF-1beta , HNF-3alpha , HNF-3beta , and/or HNF-4 expression vectors. A pGL3-basic vector was used as a negative control and a pGL3-control vector containing SV40 promoter and enhancer sequences was used as a positive control in all transfection experiments. The pRL-TK vector was also included in all transfections as an internal control for transfection efficiency as monitored by the Promega dual-luciferase reporter assay system. Cells (1-1.5 × 106) were plated on 60-mm tissue culture dishes or six-well plates and incubated for 24 h before transfection. Duplicate or triplicate dishes were transfected using the FuGENE 6 transfection reagent or the calcium phosphate-DNA coprecipitation method. For the calcium phosphate method, the cells were shocked with 15% glycerol 24 h after transfection. Cells were harvested 48 h after transfection. Luciferase activity was detected on the Turner Designs luminometer (model TD-120/20, Promega).

Preparation of double-strand oligonucleotides and labeling. Oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer in the Nucleic Acid Chemistry Laboratory, Biotechnology Center, Washington University School of Medicine. The potential HNF-1-binding sequence corresponds to the alpha 1-AT promoter sequence from -87 to -52 (5'-ATAACTGGGGTGA-CCTTGGTTAATATTCACCAGCAG-3'). The potential HNF-4-binding sequence corresponds to the sequence from -128 to -93 (5'-ATCCAGCCAGTGGACTTAGCCCCGTTTG-3') with respect to the downstream transcriptional start site. The HNF-3 consensus sequence corresponds to 5'-GCCCATTGTTTGTTTTAAGCC-3', and the HNF-4 consensus sequence corresponds to 5'-GGAAAGGTCCAAAGGGCGCCTTG-3'. The oligonucleotides were purified by denaturing PAGE, annealed at 65°C for 1 h, allowed to cool slowly at room temperature, and then labeled using [alpha -32P]dCTP and the Klenow fragment of DNA polymerase I. Radiolabeled oligonucleotides were separated from free nucleotide by nondenaturing PAGE.

EMSA. Complementary oligonucleotide probes for the HNF-1 and HNF-4 sequences in the alpha 1-AT promoter as well as consensus HNF-1, HNF-3, and HNF-4 sequences were labeled with [32P]dCTP by the 3'-end filling reaction, and 2 × 104 counts/min were incubated for 20-40 min at room temperature with nuclear extracts from Hep G2, Caco-2, H441, and human respiratory epithelial cells (25). The nuclear extracts from H441, Hep G2, and Caco-2 cells were prepared according to the protocol described by Dignam et al. (11). Nuclear extracts from HeLa cells (HeLa Scribe nuclear extract, in vitro transcription grade) were purchased from Geneka Biotechnology (Montreal, PQ, Canada). Nuclear extracts from human respiratory epithelial cells in primary culture, also prepared by the method of Dignam et al., were kindly provided by Dwight Look (St. Louis, MO). Nuclear extracts were resuspended in buffer so that a 10-µl reaction volume contained a final concentration of 60 mM KCl, 25 mM HEPES, pH 7.6, 1 mM dithiothreitol, 0.1 mM EDTA, 7.5% glycerol, 1 µg of poly(dI-dC), and 2% polyvinyl alcohol. Unlabeled oligonucleotide in molar excess was used in designated experiments. Nuclear extract dialysis buffer and BSA were used as negative controls. Rabbit polyclonal anti-HNF-1alpha TC 284 (kindly provided by M. Yaniv and M. Pontoglio, Paris, France), anti-HNF-1beta [kindly provided by G. Ryffel, Essen, Germany (35)], anti-HNF-3alpha and HNF-3beta (kindly provided by R. H. Costa, Urbana, IL), and anti-HNF-4 (kindly provided by I. Talianidis, Crete) were used in designated experiments by incubation with nuclear extracts for 2 h at 4°C before addition of radiolabeled oligonucleotide probe. The products were analyzed on 5% polyacrylamide-2.5% glycerol gels cast in 0.5× Tris-borate-EDTA.

RT-PCR for analysis of RNA levels. Total RNA from H441, Hep G2, and Caco-2 cells was isolated with the RNeasy kit (Qiagen, Hilden, Germany) and then digested with RQ1 RNase-free DNase (Promega). Poly(A)+ RNA was isolated using oligo(dT) cellulose column chromatography (32). Poly(A)+ RNA from human respiratory epithelial cells was kindly provided by Drs. Theresa Joseph and Dwight Look. The RNA was then subjected to RT-PCR using the Access RT-PCR system (Promega). The primers for amplification were based on previous studies (10, 12): 5'-GAAAGCAACGGGAGATCCTCCGAC-3' (sense) and 5'-CCTCCACTAAGGCCTCCCTCTCTTCC-3' (antisense) for HNF-1beta , 5'-GTAGACAGTAGGGGCTC-3' (sense) and 5'-GGGGAATCCTTTAAACGG-3' (antisense) for HNF-3alpha , 5'-GCCTGAGCCGCGCTCGGGAC-3' (sense) and 5'-GGTGCAGGGTCCAGAAGGAG-3' (antisense) for HNF-3beta , 5'-CTTCCTTCTTCATGCCAG-3' (sense) and 5'-ACACGTCCCCATCTGAAG-3' (antisense) for HNF-4, 5'-TCACGTCTAGAACAGTGAATCGAC-3 (sense) and 5-GTGGGCTGCAGTACCAGCTCAACC-3' (antisense) for alpha -AT, and 5'-AGGGCTGAGTGTTCTGGGATTTC-3' (sense) and 5'-GGTTACGGCAGCACTTTTATTTTT-3' (antisense) for beta -actin. Minimal modifications were applied to the manufacturer's instructions for the conditions: annealing was done at 60-65°C depending on melting temperature of oligonucleotides, the concentration of MgSO4 was 1 mM, and 35 cycles were used for amplifications. PCR products were separated on 1% agarose gels and visualized by ethidium bromide staining.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of alpha 1-AT in the human pulmonary epithelial cell line H441. RT-PCR analysis showed the presence of alpha 1-AT RNA in the H441 cell line as well as in Hep G2 and Caco-2 cells (Fig. 1). This data indicated that we could use the H441 cell line as a model for expression of the alpha 1-AT gene in pulmonary epithelium.


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 1.   Expression of alpha 1-antitrypsin (alpha 1-AT) in H441, Hep G2, and Caco-2 cells. Total cellular poly(A)+ RNA from H441, Hep G2, and Caco-2 cells was subjected to RT-PCR analysis for alpha 1-AT and beta -actin.

To determine whether there were cis-acting regulatory elements within the upstream flanking region for expression of the alpha 1-AT gene in H441 cells and whether these differed from those responsible for expression in Caco-2 and Hep G2 cells, we used seven alpha 1-AT-luciferase fusion plasmids differing in the length of the alpha 1-AT 5'-flanking sequence upstream of the luciferase coding sequence (Fig. 2). We examined the expression of these plasmids in H441, Caco-2 and Hep G2 cells. The results show that the general localization of cis-acting elements in H441 cells is similar to that in Caco-2 and Hep G2 cells. There is a drop in expression on deletion from -991 to -661, -490 to -270, -270 to -137, and -137 to -2, indicating the presence of cis-acting elements in these regions. The major positive elements were in the two most proximal regions. We decided to first examine the most proximal region, -137 to -2, in H441 cells in more detail. Previous studies showed that this region contains most of the elements responsible for tissue-specific regulation of transcription (17 and references therein). Moreover, all the potential HNF-1-, HNF-4-, and HNF-3-binding sequences are found in this proximal promoter region.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Expression in H441, Hep G2, and Caco-2 cells of alpha 1-AT-luciferase fusion plasmids carrying progressive deletions of 5'-flanking sequences. Left: schematic drawings of deletions. Right: results, reported as relative luciferase activity, which represents percent maximal activity compared with activity of the plasmid carrying the longest 5'-flanking sequence, assumed to give 100% luciferase activity. Cells were cotransfected with alpha 1-AT promoter-luciferase fusion plasmids (7.5 µg) and the pRL-TK control plasmid (0.1 µg). For H441 cells, the FuGENE 6 transfection reagent was used. The calcium phosphate-DNA coprecipitation method was used for Hep G2 and Caco-2 cells. After 48 h, firefly and Renilla luciferase activities were measured and are reported as means ± SD for 3 separate determinations.

Binding of nuclear proteins from H441 cells to the proximal alpha 1-AT promoter. Because our previous studies indicated that HNF-1 and HNF-4 and cis-acting HNF-1- and HNF-4-binding elements in the proximal upstream flanking region of the alpha 1-AT gene play a major role in expression of alpha 1-AT in one extrahepatic site, intestinal epithelium, we first used EMSA to determine whether HNF-1 and HNF-4 are expressed in H441 cells and whether they bind to the HNF-1 (-87 to -52)- or HNF-4 (-128 to -93)-binding regions of the proximal alpha 1-AT promoter (Fig. 3). The results show that the HNF-1-binding region (-87 to -52) forms a single complex with nuclear extracts from H441 cells compared with two complexes present in Hep G2, undifferentiated Caco-2 (day 1), and differentiated Caco-2 (day 4) cells. Our previous studies showed that the more slowly migrating complex corresponds to HNF-1alpha and the more rapidly migrating complex corresponds to HNF-1beta (17). These previous studies also showed an increase in the relative proportion of HNF-1alpha compared with HNF-1beta during differentiation of Caco-2 cells. The single complex present in H441 cells appears to correspond to HNF-1beta . These data also show a marked increase in the relative proportion of HNF-1alpha compared with HNF-1beta in Hep G2 compared with Caco-2 cells and a trace of the HNF-1beta complex in HeLa cells that do not express alpha 1-AT.


View larger version (79K):
[in this window]
[in a new window]
 
Fig. 3.   Trans-acting nuclear proteins from H441 cells, Hep G2 cells, undifferentiated Caco-2 cells [day 1 (d1)], differentiated Caco-2 cells [day 4 (d4)], and HeLa cells that bind to the hepatocyte nuclear factor (HNF)-1 (-87 to -52)- and HNF-4 (-128 to -93)-binding regions of the proximal alpha 1-AT promoter and to the HNF-3 and HNF-4 consensus sequences as assessed by electrophoretic mobility shift assay (EMSA). Nuclear extracts were reacted with radioactive probe, and reaction products were subjected to PAGE. Relative migration of specific DNA-binding proteins is indicated on left for HNF-1alpha , HNF-1beta , HNF-3, and HNF-4.

The HNF-4-binding region (-128 to -93) forms a single complex with Hep G2 and Caco-2 cells but does not form a complex with nuclear proteins from H441 or HeLa cells (Fig. 3). This complex is much more abundant in Caco-2 than in Hep G2 cells and decreases during differentiation of Caco-2 cells. Because it comigrates with the complex formed with the HNF-4 consensus sequence and migrates faster than the complex formed with the HNF-3 consensus sequence, this complex is probably formed by HNF-4, not HNF-3.

The HNF-3 consensus sequence forms a single complex in H441, Hep G2, Caco-2, and HeLa cells. These data indicate that HNF-3 is present in H441, Hep G2, and Caco-2 cells but does not bind to the only two potential sites for HNF-3 binding in the proximal promoter of the alpha 1-AT gene. Together, these initial EMSA studies suggest that HNF-1beta , but not HNF-3, binds to the proximal alpha 1-AT promoter in H441 cells. There is no evidence for HNF-4 in H441 cells.

To determine the specificity of each of these complexes, EMSA was done in the absence or presence of unlabeled specific and unrelated oligonucleotide competitors and antibodies. The single complex formed in H441 cells with the HNF-1-binding region of the alpha 1-AT promoter (-87 to -52) is blocked by unlabeled -87 to -52 oligonucleotide but not by unlabeled glucocorticoid response element (GRE) oligonucleotide (Fig. 4A). It is also blocked by antibody to HNF-1beta (Fig. 4B). Our previous studies showed that the more slowly migrating complex formed with the HNF-1-binding region of the alpha 1-AT promoter in Hep G2 and Caco-2 cells is supershifted by antibody to HNF-1alpha (17). In Fig. 4B, we also examined nuclear extracts from primary cultures of human respiratory epithelial cells and found a single complex that was supershifted by antibody to HNF-1beta . These results indicated that HNF-1beta also binds to the proximal alpha 1-AT promoter in human airway epithelium in vivo. It is not clear to us at this time why the single complex in human respiratory epithelial cells is slightly slower in migration than that in H441 cells.


View larger version (83K):
[in this window]
[in a new window]
 
Fig. 4.   Specificity of trans-acting nuclear proteins from H441, Hep G2, Caco-2, and human respiratory epithelial (Hu resp epi) cells in primary culture that bind to the HNF-1 (-87 to -52)- and HNF-4 (-128 to -93)-binding regions of the proximal alpha 1-AT promoter and to the HNF-3 consensus sequence as assessed by EMSA. Reactions were carried out and analyzed as described in MATERIALS AND METHODS. A: 5 µg of nuclear extracts were preincubated with 2 µl of anti-HNF-1alpha or anti-alpha 1-AT antibody before the reaction with labeled probe was initiated. Unlabeled alpha 1-AT HNF-1 oligonucleotide or unlabeled irrelevant glucocorticoid response element (GRE) oligonucleotide was added to separate reactions in 20-, 40- and 100-fold molar excesses. B: 1 µl of anti-HNF-1beta antibody was used. C: unlabeled alpha 1-AT HNF-4 oligonucleotide was added to separate reactions in 20- and 50-fold molar excess, and unlabeled irrelevant GRE probe was added in 50-fold molar excess. D: 1 µl of anti-HNF-3alpha , anti-HNF-3beta , and anti-alpha 1-AT antibody was used. Unlabeled HNF-3 consensus and irrelevant GRE oligonucleotides were added to separate reactions in 20-, 40-, and 100-fold molar excess. Arrows, primary band shift.

Next, we examined the specificity of complexes formed with the HNF-4-binding region of the alpha 1-AT promoter. The single complex formed with this region in Hep G2 and Caco-2 (18) cells is blocked by unlabeled -128 to -93 oligonucleotide, but not by unlabeled GRE oligonucleotide, and is supershifted by antibody to HNF-4 (Fig. 4C). A complex is not formed with this region by nuclear proteins from H441 cells.

The single complex formed by nuclear proteins from H441 cells with the HNF-3 consensus sequence is blocked by antibody to HNF-3alpha and antibody to HNF-3beta but not by antibody to alpha 1-AT (Fig. 4D). It is also blocked by unlabeled HNF-3 but not by unlabeled GRE oligonucleotide. This shows the identity and specificity of the complex formed with the HNF-3 consensus sequence by HNF-3 in H441 cells. Together with the results in Fig. 1, these data show that HNF-3 is present in H441 cells but does not bind to the only potential HNF-3-binding sites in the proximal alpha 1-AT promoter.

Role of HNF-1 and HNF-4 in expression of alpha 1-AT in H441 cells. H441 cells were compared with Hep G2 and Caco-2 cells for luciferase activity after cotransfection of the alpha 1-AT promoter (-137 to -2)-luciferase reporter plasmid and HNF-1beta expression plasmid (Fig. 5A). The results show that HNF-1beta mediates a concentration-dependent increase in luciferase activity in H441, but not in Hep G2 or Caco-2, cells. In contrast, HNF-1alpha mediates increases in luciferase activity in all three cell types, albeit to a significantly greater extent in H441 than in the other two cell types (Fig. 5B). The role of HNF-1 in expression of alpha 1-AT in H441 cells was also shown by comparing the luciferase activity of the alpha 1-AT promoter (-137 to -2)-luciferase reporter plasmid with that of the corresponding Mut-2 plasmid, mutated in the HNF-1-binding site. There was a marked decrease in the luciferase activity of the Mut-2 plasmid (data not shown).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of HNF-1beta and HNF-1alpha on expression of the alpha 1-AT gene in H441, Caco-2, and Hep G2 cells using the alpha 1-AT proximal promoter (-137 to -2)-luciferase fusion plasmid. Cells were cotransfected with alpha 1-AT (-137 to -2)-luciferase reporter plasmid (7.5 µg) and pRL-TK plasmid (0.1 µg) in the absence or presence of HNF-1beta (A) or HNF-1alpha (B) expression plasmids in the amounts indicated. H441 cells were transfected with the FuGENE 6 reagent, and Hep G2 and Caco-2 cells were transfected by the calcium phosphate-DNA coprecipitation method. After 48 h, firefly and Renilla luciferase activities were measured. Relative luciferase activity represents normalized luciferase activity of each condition compared with normalized luciferase activity in cells transfected with the alpha 1- AT promoter alone. Values are means of triplicate determinations at each point.

Next, we examined the effect of HNF-1beta and HNF-4 together. Our previous studies showed that HNF-1alpha and HNF-4 have a synergistic effect on alpha 1-AT expression in Hep G2 and Caco-2 cells (17). The results in Fig. 6 are very similar to those in Fig. 5, showing that HNF-1alpha alone mediates an increase in luciferase activity in all three cell types but HNF-1beta alone mediates a significant increase only in H441 cells. HNF-4 by itself only mediates a two- to threefold increase in the three cell types. The combination of HNF-1alpha and HNF-4 has an additive effect on luciferase activity in Hep G2 cells, a marked synergistic effect in Caco-2 cells, but neither additive nor synergistic effects in H441 cells. The combination of HNF-1beta and HNF-4 is also synergistic in Caco-2 cells but neither additive nor synergistic in Hep G2 or H441 cells. These data show that HNF-1beta plays a unique role in alpha 1-AT expression in H441 cells and that HNF-4 has minimal activity in H441 cells in the absence or presence of HNF-1alpha or HNF-1beta .


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of HNF-1beta together with HNF-4 on expression of the alpha 1-AT gene in H441, Caco-2, and Hep G2 cells. Cells were cotransfected with the alpha 1-AT (-137 to -2)-luciferase reporter plasmid (7.5 µg) and pRL-TK plasmid (0.1 µg) in the absence or presence of HNF-1alpha expression plasmid (0.4 µg), HNF-1beta expression plasmid (0.4 µg), HNF-4 expression plasmid (0.4 µg), or a combination of these expression plasmids. Experimental protocol was otherwise identical to that described Fig. 5 legend. Values are means ± SD for triplicate determinations of each condition.

Effects of HNF-3 on alpha 1-AT gene expression in H441 cells. Previous studies indicated that HNF-3 plays a dominant role in gene expression in H441 and pulmonary epithelial cells (3-5, 28). Moreover, HNF-3 has been shown to interact with HNF-1 and HNF-4 and to bind to a cis-acting sequence element that overlaps with the element that binds HNF-1 and HNF-4 (8, 13-15, 22, 23, 33). Therefore, we examined the role of HNF-3 in alpha 1-AT gene expression in H441 compared with Hep G2 and Caco-2 cells (Fig. 7). The results show that HNF-3 by itself has no effect on luciferase activity in H441 cells. Moreover, HNF-3 does not mediate a significant effect on alpha 1-AT gene expression in H441 cells when added together with HNF-1alpha or HNF-1beta . HNF-3 also has no effect by itself on luciferase activity in Hep G2 and Caco-2 cells but does mediate a modest upregulatory effect when added together with HNF-1alpha in Hep G2 and Caco-2 cells. The lack of effect could not be attributed to saturation by endogenous HNF-3 in the nuclear extract, because there was no significant reduction in luciferase activity in H441 cells transfected with the alpha 1-AT (-137 to -2)-luciferase promoter mutated in the HNF-3-binding site compared with the same plasmid without the mutation (data not shown). These data indicate that HNF-3 has no effect on alpha 1-AT gene expression in pulmonary epithelial cells. HNF-3 does have an additive effect with HNF-1alpha in Hep G2 and Caco-2 cells, even though it does not bind to the proximal alpha 1-AT promoter, implying that this effect involves an interaction with HNF-1alpha or with a cofactor necessary for HNF-1 activity.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of HNF-3 together with HNF-1 on expression of the alpha 1-AT gene in H441, Hep G2, and Caco-2 cells. The protocol was identical to that described in Fig. 5 legend. HNF-3alpha and HNF-3beta plasmids were used at 0.1 µg, and HNF-1alpha and HNF-1beta plasmids were used at 0.2 µg.

Expression of alpha 1-AT and HNF-1beta in primary cultures of human respiratory epithelial cells. To examine the possibility that HNF-1beta is also involved in expression of the alpha 1-AT gene in another respiratory epithelial cell system, we used RT-PCR analysis of human respiratory epithelial cells in primary culture (Fig. 8). The results show the presence of alpha 1-AT and HNF-1beta RNA in the cultured cells, as well as in H441, Hep G2, and Caco-2 cells. The results also show HNF-3alpha and HNF-3beta , but not HNF-4, RNA in primary cultures, demonstrating that the repertoire of these transcription factors expressed in H441 cells faithfully reflects respiratory epithelium in vivo. Taken together with data from the EMSA (Fig. 4B), these results provide further evidence for the unique involvement of HNF-1beta in alpha 1-AT gene expression in respiratory epithelial cells.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 8.   Expression of alpha 1-AT, HNF-1beta , HNF-3alpha , HNF-3beta , and HNF-4 in cultured human respiratory epithelial cells. Total cellular RNA from H441, Hep G2, and Caco-2 cells and poly(A)+ RNA from primary cultures of human respiratory epithelial cells were subjected to RT-PCR analysis for alpha 1-AT, HNF-1beta , HNF-3alpha , HNF-3beta , HNF-4, and beta -actin.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although alpha 1-AT has always been considered a hepatic-derived plasma protein, there is abundant evidence that it is synthesized in extrahepatic sites by mononuclear phagocytes and epithelial cells (reviewed in Ref. 28). Interestingly, extrahepatic synthesis of alpha 1-AT is particularly characteristic of the human compared with the mouse and rat. In fact, early studies of transgenic mice, engineered to express human alpha 1-AT by using as transgene a genomic fragment encompassing the coding region and most of its 5'-flanking region, showed expression of human alpha 1-AT, but not endogenous mouse alpha 1-AT, in many extrahepatic tissues (19). More recent studies have shown that alpha 1-AT is synthesized in human airway epithelial cells in primary culture, cell lines, and human lung tissue in situ (9, 21, 22). Airway epithelium is likely to be a particularly important site of synthesis for alpha 1-AT, because it is the major physiological inhibitor of neutrophil elastase, cathepsin G, and proteinase 3, neutrophil proteases that can degrade the connective tissue matrix of the lung in vivo, and because alpha 1-AT deficiency predisposes to destructive lung disease/emphysema (20). However, the detection of alpha 1-AT gene expression in a cell line and primary cultures derived from respiratory epithelium here and in previous studies (9, 22) does not by itself indicate that this expression is physiologically relevant. Even the detection of alpha 1-AT mRNA in human respiratory epithelium by in situ hybridization analysis (21) does not ensure that a physiological function for alpha 1-AT derived from this source as opposed to the liver, in which expression levels are much higher. Previous studies showed that the allotype of alpha 1-AT in plasma converts to that of the donor after orthotopic liver transplantation (1, 16), indicating that plasma alpha 1-AT is predominantly derived from liver. Whether extrahepatic expression is physiologically relevant will be definitively addressed only if there is correction of a functional abnormality by tissue-specific transgenic expression of alpha 1-AT in an alpha 1-AT-knockout mouse.

In this study, we examined the possibility that expression of alpha 1-AT in human pulmonary epithelial cells involved transcriptional mechanisms similar to those at play in intestinal epithelial cells and/or hepatocytes. Our previous studies showed that the synergistic action of HNF-1alpha and HNF-4 plays a prominent role in alpha 1-AT transcription in enterocytes and hepatocytes and in the mechanism by which the alpha 1-AT gene is upregulated during differentiation of enterocytes from crypt to villous tip (26). Previous studies from other laboratories have implicated HNF-3 and thyroid transcription factor-1 in gene expression in pulmonary epithelium (3-5, 30). To our surprise, the results showed that alpha 1-AT gene expression in pulmonary epithelial cells is predominantly driven by HNF-1 and not at all by HNF-3. Even more surprising, HNF-1beta is responsible for activating alpha 1-AT gene expression in pulmonary epithelial cells, even though it has no effect on the alpha 1-AT gene in intestinal epithelial cells or hepatocytes. HNF-1beta is highly homologous to HNF-1alpha , particularly in the NH2-terminal DNA-binding region, but diverges within the COOH-terminal activation domain (7). There is relatively limited information about the function of HNF-1beta , except that it is expressed in dedifferentiated cells and somatic cell hybrids that have lost the ability to express liver-specific genes and lack the transcription factors HNF-1alpha and HNF-4 (2). HNF-1beta is also known to be expressed in some tissues that do not express HNF-1alpha , including thymus, testis, ovary, and lung (2, 7), but this is the first report that we can find in the literature of a specific transcriptional role for HNF-1beta in one of these tissues. It is also noteworthy that HNF-1beta activates the alpha 1-AT gene, a gene characteristic of the differentiated hepatocyte, in lung cells, but not in hepatocytes or enterocytes. These data imply that there are cell type-specific mechanisms for the action of HNF-1beta and militate against it being merely a part of dedifferentiation from the hepatic phenotype. The fact that HNF-4 does not appear to mediate a synergistic effect with HNF-1beta on alpha 1-AT gene expression in lung cells, even though it does mediate a synergistic effect with HNF-1alpha in liver and intestinal cells, also implies cell type specificity in the role of HNF-1beta on the alpha 1-AT gene in lung cells.

The results also indicate that HNF-3alpha and HNF-3beta do not directly activate alpha 1-AT gene expression in lung, intestinal, or liver cells. This is somewhat surprising, because HNF-3 appears to play a major role in gene expression in differentiated pulmonary epithelial cells, and there are two potential HNF-3-binding sites within the proximal promoter of the alpha 1-AT gene. We did find, however, that HNF-3alpha and HNF-3beta have an additive effect with HNF-1alpha on alpha 1-AT gene expression in Hep G2 and Caco-2 cells. Previous work has shown that HNF-3 interacts with HNF-1alpha in regulation of the liver-specific trans-activation of aldolase-beta , but in this case the interaction is antagonistic (14). Because HNF-3alpha and HNF-3beta do not bind to the proximal promoter of the alpha 1-AT gene, their additive effect must involve interaction with HNF-1alpha or a cofactor necessary for transcriptional activation by HNF-1alpha .


    ACKNOWLEDGEMENTS

The authors are indebted to M. Maksin for preparation of the manuscript.


    FOOTNOTES

The studies were supported in part by National Institutes of Health Grants HL-37784 and DK-52526.

Address for reprint requests and other correspondence: D. Perlmutter, Dept. of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213-2583 (E-mail: perldav{at}chplink.chp.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.

10.1152/ajplung.00271.2001

Received 25 September 2001; accepted in final form 21 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alper, CA, Raum D, Awdeh ZL, Petersen BH, Taylor PD, and Starzl TE. Studies of hepatic synthesis in vivo of plasma proteins, including orosomucoid, transferrin, alpha 1-antitrypsin, C8, and factor B. Clin Immunol Immunopathol 16: 84-89, 1980[ISI][Medline].

2.   Baumhueter, S, Courtois G, and Crabtree GR. A variant nuclear protein in dedifferentiated hepatoma cells binds to the same functional sequences in the beta -fibrinogen gene promoter as HNF-1. EMBO J 7: 2485-2491, 1988[Abstract].

3.   Bingle, CD, Hackett BP, Moxley M, Longmore W, and Gitlin JD. Role of hepatocyte nuclear factor-3alpha and hepatocyte nuclear factor-3beta in Clara cell secretory protein gene expression in the bronchiolar epithelium. Biochem J 308: 197-202, 1995[ISI][Medline].

4.   Braun, H, and Suske G. Combinatorial action of HNF3 and Sp family transcription factors in the activation of the rabbit uteroglobin/CC10 promoter. J Biol Chem 273: 9821-9828, 1998[Abstract/Free Full Text].

5.   Bruno, MD, Bohinski RJ, Huelsman KM, Whitsett JA, and Korfhagen TR. Lung cell-specific expression of the murine surfactant protein A (SP-A) gene is mediated by interactions between the SP-A promoter and thyroid transcription factor-1. J Biol Chem 270: 6531-6536, 1995[Abstract/Free Full Text].

6.   Carrell, R, Lomas D, Stein P, and Whisstock J. Dysfunctional variants and the structural biology of the serpins. Adv Exp Med Biol 425: 207-222, 1997[ISI][Medline].

7.   Cereghini, S. Liver-enriched transcription factors and hepatocyte differentiation. FASEB J 10: 267-282, 1996[Abstract/Free Full Text].

8.   Chaya, D, Fougere-Deschatrette C, and Weiss M. Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines. Mol Cell Biol 17: 6311-6320, 1997[Abstract].

9.   Cichy, J, Potempa J, and Travis J. Biosynthesis of alpha 1-proteinase inhibitor by human lung-derived epithelial cells. J Biol Chem 272: 8250-8255, 1997[Abstract/Free Full Text].

10.   Coffinier, C, Thepot D, Babinet C, Yaniv M, and Bara J. Essential role for the homeoprotein vHNF1/HNF1beta in visceral endoderm differentiation. Development 126: 4785-4794, 1999[Abstract/Free Full Text].

11.   Dignam, JD, Lebovitz RM, and Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475-1489, 1983[Abstract].

12.   Duncan, SA, Nagy A, and Chan W. Murine gastrulation requires HNF-4 regulated gene expression in the visceral endoderm: tetraploid rescue of Hnf-4 (-/-) embryos. Development 124: 279-287, 1997[Abstract/Free Full Text].

13.   Duncan, SA, Navas MA, Dufort D, Rossant J, and Stoffel M. Regulation of a transcription factor network required for differentiation and metabolism. Science 281: 692-695, 1998[Abstract/Free Full Text].

14.   Gregori, C, Kahn A, and Pichard AL. Activity of the rat liver-specific aldolase B promoter is restrained. Nucleic Acids Res 22: 1242-1246, 1994[Abstract].

15.   Harnish, DC, Malik S, Kilbourne E, Costa R, and Karathanasis SK. Control of apolipoprotein AI gene expression through synergistic interactions between hepatocyte nuclear factors 3 and 4. J Biol Chem 271: 13621-13628, 1996[Abstract/Free Full Text].

16.   Hood, JM, Koep LJ, Peters RL, Schroter GP, Weil R, Redeker AG, and Starzl TE. Liver transplantation for advanced liver disease with alpha 1-antitrypsin deficiency. N Engl J Med 302: 272-275, 1980[ISI][Medline].

17.   Hu, C, and Perlmutter DH. Regulation of alpha 1-antitrypsin gene expression in human intestinal epithelial cell line Caco-2 by HNF1alpha and HNF4. Am J Physiol Gastrointest Liver Physiol 276: G1181-G1194, 1999[Abstract/Free Full Text].

18.   Huber, R, and Carrell RW. Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins. Biochemistry 117: 48-53, 1990.

19.   Kelsey, GD, Povey S, Bygrave AE, and Lovell-Badge RH. Species and tissue-specific expression of human alpha 1-antitrypsin in transgenic mice. Genes Dev 1: 161-170, 1987[Abstract].

20.   Knight, KR, Burdon JG, Cook L, Brenton S, Ayad M, and Janus ED. The proteinase-antiproteinase theory of emphysema: a speculative analysis of recent advances into the pathogenesis of emphysema. Respirology 2: 91-95, 1997[Medline].

21.   Koopman, P, Povey S, and Lovell-Badge RH. Widespread expression of human alpha 1-antitrypsin in transgenic mice revealed by in situ hybridization. Genes Dev 3: 16-25, 1989[Abstract].

22.   Ktistaki, E, and Talianidis I. Modulation of hepatic gene expression by hepatocyte nuclear factor 1. Science 277: 109-112, 1997[Abstract/Free Full Text].

23.   Levinson-Dushnik, M, and Benvenisty N. Involvement of hepatocyte nuclear factor 3 in endoderm differentiation of embryonic stem cells. Mol Cell Biol 17: 3817-3822, 1997[Abstract].

24.   Look, DC, Rapp SR, Keller BT, and Holtzman MJ. Selective induction of intercellular adhesion molecule-1 by interferon-gamma in human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 263: L79-L87, 1992[Abstract/Free Full Text].

25.   Mendel, DB, and Crabtree GR. HNF-1, a member of a novel class of dimerizing homeodomain proteins. J Biol Chem 266: 677-680, 1991[Free Full Text].

26.   Molmenti, E, Perlmutter DH, and Rubin D. Cell-specific expression of alpha 1-antitrypsin in intestinal epithelial cells. J Clin Invest 92: 2022-2034, 1993[ISI][Medline].

27.   O'Reilly, MA, Weaver TE, Ilot-Matias TJ, Sarin VK, Gadzar AF, and Whitsett JA. In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors. Biochim Biophys Acta 1101: 140-148, 1989.

28.   Perlmutter, DH. alpha 1-Antitrypsin deficiency. In: Schiff's Diseases of the Liver (8th ed.), edited by Schiff ER, Sorrell MF, and Maddrey WC.. Philadelphia, PA: Lippincott-Raven, 1999, p. 1131-1150.

29.   Perlmutter, DH, Cole FS, Kilbridge P, Rossing TH, and Colten HR. Expression of the alpha 1-proteinase inhibitor gene in human monocytes and macrophages. Proc Natl Acad Sci USA 82: 795-799, 1985[Abstract].

30.   Ray, MK, Chen CY, Schwartz RJ, and DeMayo FJ. Transcriptional regulation of a mouse Clara cell-specific protein (mCC10) gene by the NKx transcription factor family members thyroid transcription factor 1 and cardiac muscle-specific homeobox protein (CSX). Mol Cell Biol 16: 2056-2064, 1996[Abstract].

31.   Rottman, JN, and Gordon JI. Comparison of the patterns of expression of rat intestinal fatty acid binding protein/human growth hormone fusion genes in cultured intestinal epithelial cell lines and in the gut epithelium of transgenic mice. J Biol Chem 268: 11994-12002, 1993[Abstract/Free Full Text].

32.   Sambrook, J, Fritsch EF, and Maniatis T. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989, p. 125-126.

33.   Vallet, V, Antoine B, Chafey P, Vandewalle A, and Kahn A. Overproduction of a truncated hepatocyte nuclear factor 3 protein inhibits expression of liver-specific genes in hepatoma cells. Mol Cell Biol 15: 5453-5460, 1995[Abstract].

34.   Venembre, P, Boutten A, Seta N, Dehoux MS, Crestani B, Aubier M, and Durand G. Secretion of alpha 1-antitrypsin by alveolar epithelial cells. FEBS Lett 346: 171-174, 1994[ISI][Medline].

35.   Wild, W, Von Strandmann EP, Nastos A, Senkel S, Lingott-Frieg A, Bulman M, Bingham C, Ellard S, Hattersley A, and Ryffel GU. The mutated human gene encoding hepatocyte nuclear factor 1beta inhibits kidney formation in developing Xenopus embryos. Proc Natl Acad Sci USA 97: 4695-4700, 2000[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 282(4):L757-L765
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (8)
Google Scholar
Articles by Hu, C.
Articles by Perlmutter, D. H.
Articles citing this Article
PubMed
PubMed Citation
Articles by Hu, C.
Articles by Perlmutter, D. H.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online