Glucocorticoid-dependent uteroglobin synthesis and uteroglobulin mRNA levels in rabbit lung explants cultured in vitro

In rabbit lung explants cultured in vitro in a synthetic medium, the synthesis of the protein uteroglobin decayed progressively becoming virtually undetectable between 24-48 h of culture. Addition of glucocorticoids to the medium maintained the synthesis of uteroglobin. This glucocorticoid effect was dose-dependent with optima at about 0.1 microM and 1 microM for dexamethasone and cortisol respectively. Estradiol, progesterone, triiodothyronine, insulin or 10% calf serum added to the medium were ineffective in maintaining uteroglobin synthesis. Actinomycin D (10 micrograms/ml) added to the medium inhibited the effect of cortisol on uteroglobin synthesis. After 24 h of culture, both the relative levels of uteroglobin mRNA, measured by molecular hybridization, and uteroglobin synthesis were correlatively higher (up to 10-fold) in glucocorticoid-treated than in control explants.     

Glucocorticoid and developmental regulation of uteroglobin synthesis in rabbit lung.

The synthesis of uteroglobin in rabbit lung was studied after the administration of glucocorticoids to intact adult animals as well as during the late stages of rabbit development. The synthesis of uteroglobin was compared with levels of translatable uteroglobin mRNA in the lung. Uteroglobin synthesis was determined both by incorporation of [25S]methionine into the protein by lung explants incubated in vitro and by radioimmunoassay measurements of uteroglobin concentration in lung. Lung poly(A)-containing mRNA, isolated by oligo(dT)--cellulose chromatography, was translated in cell-free systems and the activity of uteroglobin mRNA was determined after immunoprecipitation. Dexamethasone administration increased about 2-fold the synthesis of lung uteroglobin compared with the controls. The effect of cortisol was more moderate. Both glucocorticoids did not affect the degradation rate of lung uteroglobin, but produced increases in the translatable levels of uteroglobin mRNA parallel to those observed for uteroglobin synthesis. During the late stages of rabbit development, both the synthesis of lung uteroglobin and the translatable levels of its mRNA increase in parallel about 12-fold in a biphasic fashion. A first increase occurred between 2 days before and 2 days after birth. Starting at 5 days of age, there was a second increase in both parameters, which at 12 days of age reached values close to those observed in adult rabbits. Our results suggest that the rate of lung uteroglobin synthesis could be mainly determined by the translatable levels of its mRNA.     

Uteroglobin mRNA from the lung of the hare (Lepus capensis): cell-free translation and properties

Poly(A)-containing RNA was obtained from the lung of hares (Lepus capensis) and uteroglobin mRNA was characterized by cell-free translation and molecular hybridization to a rabbit uteroglobin cDNA probe. In the cell-free system, hare uteroglobin mRNA was preferentially translated as compared to the whole lung mRNA and it directed the synthesis of a precursor, preuteroglobin, containing about twenty additional amino acids. Hare uteroglobin mRNA was about 40 nucleotides larger than the homologous rabbit one, as analyzed by electrophoresis on agarose gel. Thermal stability of the hybrids formed between rabbit or hare uteroglobin mRNAs and the rabbit cDNA probe indicated differences in the nucleotide sequence of both mRNAs. The levels of uteroglobin mRNA and uteroglobin synthesis in lung are about two-fold higher in hare than in rabbit lung.     

Tissue-specific expression, hormonal regulation and 5''-flanking gene region of the rat Clara cell 10 kDa protein: comparison to rabbit uteroglobin.

The amino acid sequence of rat Clara Cell 10 kDa secretory protein (CC10) shows 55% identity to rabbit uteroglobin. In order to define the relationship between rat CC10 and rabbit uteroglobin in detail, the tissue-specific expression and hormonal regulation of rat CC10 mRNA was analyzed. We report that like rabbit uteroglobin, rat CC10 mRNA is expressed in lung and esophagus, as well as in uteri of estrogen- and progesterone-treated females. Expression of CC10 mRNA in lung is regulated by glucocorticoids. The similarity in expression pattern of rat CC10 mRNA and rabbit uteroglobin mRNA is reflected by a striking similarity in the 5'-flanking regions of the two genes. Despite this overall similarity, two regions of 0.3 kb and 2.1 kb are absent in the rat CC10 upstream gene region. The larger region includes a cluster of hormone receptor binding sites, believed to be responsible for differential regulation of rabbit uteroglobin by glucocorticoids and progesterone. Thus, while the sequence identities in the coding and 5'-flanking regions point towards a common ancestor for the uteroglobin and CC10 gene, later events (deletions/insertions) might have caused species-specific differences in their regulation.     

Testosterone induces the expression of the uteroglobin gene in rabbit epididymis.

Uteroglobin was characterized in the rabbit epididymis by radioimmunoassay and electrophoretic determinations, as well as by analysis of its mRNA by means of 'Northern blot' and nuclease-S1 mapping. Treatment of sexually immature rabbits with testosterone during 5 days increased up to 8-fold the concentrations of both uteroglobin and its mRNA in the epididymis. The amounts of beta-tubulin mRNA, measured as reference, remained unchanged after the hormonal treatment. The synthesis of uteroglobin occurred mainly in the middle region of the epididymis, progressively decreasing toward the distal part of the organ. Uteroglobin was not detected in the testis by radioimmunoassay. The results are discussed in relation to a possible role of uteroglobin in the reproductive functions.     

Isolation and characterization of uteroglobin from the lung of the hare (Lepus capensis)

Uteroglobin has been purified from hare lung by gel filtration and chromatography on carboxymethyl-cellulose. Hare uteroglobin appears homogeneous by electrophoresis under both denaturing and nondenaturing conditions. Its chemical and immunological properties as well as its ability to bind progesterone are compared to those of rabbit uteroglobin. The two proteins have the same N-terminal residue (glycine) and both lack tryptophan but differ in amino acid composition. Sodium dodecyl sulfate-gel electrophoresis shows that hare uteroglobin is composed of two subunits of identical Mr (about 7000) held together by disulfide bridges. The amino acid composition indicates a subunit composed of 65-67 residues, which is compatible with the apparent Mr observed. Thus, hare uteroglobin appears to be slightly smaller than the rabbit protein. Hare uteroglobin partially reacts with anti-rabbit uteroglobin in a radioimmunoassay and also binds progesterone, although this binding is relatively unaffected by dithiothreitol. The synthesis of hare uteroglobin in the uterus appears to be rather insensitive to ovarian steroid hormones.     

Uterine and lung uteroglobins in the rabbit: Two similar proteins with differential hormonal regulation

Previous studies have shown that several rabbit tissues contain proteins which cross-react in the radioimmunoassay for uteroglobin, a progestin-regulated protein in rabbit uterus (Torkkeli et al. (1977) Mol. Cell. Endocrinol. 9, 101–118). In the present study, a uteroglobin-like protein was purified to an apparent homogeneity from an extra-uterine tissue, rabbit lung, by successive chromatographies on hydroxyapatite, Sephadex G-75, SP-Sephadex, DEAE-cellulose and CM-cellulose. The final preparation behaved homogeneously in various polyacrylamide gel electrophoretic systems and in isoelectric focusing. The uteroglobin-like protein isolated from the lung had very similar physico-chemical and immunological properties to those of uteroglobin present in the rabbit uterine fluid. The two proteins had: (i) the same molecular weight, of approx. 13 000, with a two subunit structure (each approx. M_r 7000); (ii) identical behavior in polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions; (iii) the same isoelectric point at pH 5.4; (iv) absence of carbohydrate in the molecule; (v) very similar amino acid compositions; (vi) lack of tryptophan among the amino acids; (vii) the same N-terminal amino acid (glycine), and (viii) indistinguishable immunological characteristics. Collectively, these data strongly suggest that uterine and lung uteroglobins are identical proteins.In contrast to the induction of the uterine uteroglobin by steroids with progestational activity, the synthesis of extra-uterine uteroglobins was not affected by these steroid hormones to any major extent. In keeping with the concept that lung is a target tissue for glucocorticoid action, cortisol and dexamethasone were capable of increasing the concentration of lung uteroglobin 3-fold (from 3 to 9 μg/mg soluble protein). These compounds did not, however, alter the secretion of the uterine protein. Administration of high doses of testosterone and 5α-dihydrotestosterone elevated significantly the content of both uterine and lung uteroglobin. Only approx. one-fifth of the adult pulmonary uteroglobin levels were present in lungs of newborn rabbits indicating that developmental changes occur in the lung uteroglobin content.     

Characteristics of the purified uteroglobin-like protein from rabbit lung.

A uteroglobin-like protein was prepared from lung extracts of female rabbits by absorption to immobilized anti-uteroglobin immunoglobulin and purified to homogeneity by gel filtration on Sephacryl S-200. The final preparation is indistinguishable from uteroglobin according to its behaviour in Ouchterlony double-diffusion, polyacrylamide gel electrophoresis under denaturing and non-denaturing conditions, ultraviolet spectrum, tryptic peptide analysis, and progesterone-binding properties. Progesterone binding to the lung protein exhibits an affinity similar to that observed with authentic uteroglobin and is equally enhanced by reduction of the protein with dithiothreitol. Competition experiments with non-radioactive steroids demonstrate a similar steroid-specificity for both proteins. Progesterone binding causes a perturbation in the ultraviolet absorbance of tyrosine residues of the lung protein similar to that observed with uteroglobin. These data suggest that the proteins prepared from both sources are biochemically identical.     

Primary structure of rabbit lung uteroglobin as deduced from the nucleotide sequence of a cDNA

Double-stranded cDNA was synthesized from partially purified uteroglobin mRNA from rabbit lung. A cDNA coding for lung uteroglobin was then cloned in the plasmid pUC18 and both the nucleotide sequence and the derived amino acid sequence were determined. This allowed us to demonstrate unequivocally that uteroglobins from lung and uterus are identical proteins.     

Variable expression of the uteroglobin gene following the administration of norethisterone and its A-ring reduced metabolites

Enzyme-mediated A-ring reduction of norethisterone (NET) results in the transformation of a molecule with potent intrinsic progestational activity into neutral derivatives with estrogen-like effects. To ascertain whether these structural modifications of NET are able to modify the uteroglobin (U) gene (G) expression, a series of experiments assessing the UG products after the administration of NET and its reduced A-ring metabolites were conducted in prepubertal female rabbits. Synthesis of endometrial uteroglobin and its specific mRNA were studied in animals following the administration of NET, 5 alpha-dihydro NET,3 beta,5 alpha-tetrahydro NET and progesterone. Animals treated with either estradiol or vehicle alone served as controls. The uteroglobin content in uterine flushings and cytosols was determined by immunodiffusion and polyacrilamide gel electrophoresis techniques and by a specific double-antibody radioimmunoassay, while the U mRNA synthesis was assessed by its molecular hybridization to [alpha 32P]d-ATP uteroglobin cDNA. NET induced a significant increase of the uterine content of uteroglobin similar to that observed with progesterone with a simultaneous increase on U mRNA synthesis. On the contrary, 5 alpha-NET and 3 beta,5 alpha-NET induced very little, if any uteroglobin synthesis with a concomitantly low U mRNA production as compared with NET; thus exhibiting a similar effect to that observed in estradiol-treated animals. The overall results were interpreted as demonstrating that the enzyme mediated structural changes of NET which occur at the target organs induce variable expression of the uteroglobin gene. The data indicate that the rabbit uteroglobin gene products are suitable molecular markers to evaluate the hormonal potency of contraceptive synthetic progestins and their derivatives.     

Recombinant rabbit uteroglobin expressed at high levels in E. coli forms stable dimers and binds progesterone

In order to express uteroglobin in Escherichia coli we have constructed a DNA coding for complete mature rabbit uteroglobin by fusing genomic sequences from the second exon of the gene to an incomplete cDNA. This DNA was inserted into various positions of the polylinker cloning region of pDS expression vectors and the uteroglobin gene was expressed in E. coli by IPTG induction. Four different uteroglobin-derived proteins were produced containing 1, 3, 5 and 7 more N-terminal amino acids than the naturally occurring mature protein. The yield of soluble protein strongly increased with increasing length of the N-terminal additions. Protein and RNA analysis showed that this variation is most likely due to progressively higher translation efficiencies of the larger recombinants. UG7, the most efficiently synthesized recombinant protein, carrying seven additional N-terminal amino acids, was purified and further characterized. Like natural uteroglobin, UG7 forms a dimer and binds progesterone with an affinity indistinguishable to the natural protein. This bacterially produced protein can be used for detailed structure-function investigations of uteroglobin.     

Connective tissue activation in guinea pig lung fibroblast cultures: regulatory effects of glucocorticoids

Earlier studies showed that guinea pig lung fibroblasts in cell culture could be "activated" by naturally occurring peptides with a resultant increase in glycolysis and glycosaminoglycan formation. Such connective tissue activation (CTA) in human cell systems (synovial, cartilage, dermal) has proved a useful tool for studying the mechanisms of inflammation and dissecting the efficacy and actions of anti-inflammatory drugs. The present study examined the consequences of treating basal and activated guinea pig lung fibroblasts with glucocorticoids. The data indicate that glucocorticoids minimally suppress glycosaminoglycan (GAG) synthesis in nonactivated cultures. Further, CTA was inhibited to only a minor degree in activated lung fibroblast cultures by steroids, and even markedly supraphysiologic concentrations of glucocorticoids were not notably inhibitory. It was of interest that thiols enhanced suppression of incremental GAG synthesis by some glucocorticoids in activated lung fibroblast cultures.     

Connective tissue activation in guinea pig lung fibroblast cultures: Regulatory effects of glucocorticoids

Summary Earlier studies showed that guinea pig lung fibroblasts in cell culture could be “activated” by naturally occurring peptides with a resultant increase in glycolysis and glycosaminoglycan formation. Such connective tissue activation (CTA) in human cell systems (synovial, cartilage, dermal) has proved a useful tool for studying the mechanisms of inflammation and dissecting the efficacy and actions of anti-inflammatory drugs. The present study examined the consequences of treating basal and activated guinea pig lung fibroblasts with glucocorticoids. The data indicate that glucocorticoids minimally suppress glycosaminoglycan (GAG) synthesis in nonactivated cultures. Further, CTA was inhibited to only a minor degree in activated lung fibroblast cultures by steroids, and even markedly supraphysiologic concentrations of glucocorticoids were not notably inhibitory. It was of interest that thiols enhanced suppression of incremental GAG synthesis by some glucocorticoids in activated lung fibroblast cultures. This study was supported by USPHS Grant HL-19685.     

Changes in cytosol and nuclear progesterone receptor concentrations in the rabbit uterus and their relation to induction of progesterone-regulated uteroglobin.

To study the relation between steroid receptor concentrations and biological response, we measured cytosol and nuclear progesterone receptors from rabbit uterus under different experimental conditions, and compared receptor values with induction of uteroglobin, a progesterone-regulated protein. A 5-day progesterone treatment (1 mg/kg per day) reduced the nuclear receptor content by 40%, slightly elevated cytosol receptor levels and increased uteroglobin content 3000-fold. Estradiol and tamoxifen altered progesterone-induced changes in the receptor and uteroglobin values: cytosol and nuclear receptors rose significantly, but uteroglobin induction declined markedly, when progesterone treatment was supplemented with estradiol or tamoxifen. A 50% inhibition of progesterone action on uteroglobin synthesis was achieved with 1 μg/kg of estradiol per day. Thus under certain conditions, there is a clear disparity between steroid receptor levels and biological response.     

Clara cell 10 kDa protein (CC10): comparison of structure and function to uteroglobin

The cellular localization, functional activities and structures of rat and human Clara cell 10 kDa proteins (CC10) are compared to rabbit uteroglobin. CC10 is present exclusively in the non-ciliated cells of the surface epithelium of the pulmonary airways, whereas uteroglobin is reported to be present in the lung and reproductive organs. There is about 55% identity between the amino acid sequences of rat CC10 and either rabbit uteroglobin or human CC10. The latter two have 61% identity. Using the known structure of uteroglobin as the model, correlations between the structure and function for this group of proteins are made. Substitution of the residues for the rat and human CC10 into the structure of uteroglobin suggests that these proteins may be members of a structurally homologous family. Some of the functional differences may be due to distortion of the hydrophobic pocket in the dimeric protein and a surface hypervariability located on one contiguous helix and beta turn. Rat CC10 and rabbit uteroglobin both, nearly equally, inhibit papain and bind progesterone. Human CC10 does not inhibit papain and has markedly lower progesterone binding (4.6% of rabbit uteroglobin). Antiinflammatory activity of synthetic peptides corresponding to a homologous sequence region of uteroglobin and the two Clara cell proteins was tested. The region chosen has sequence similarity to lipocortin I. The peptides not only failed to inhibit carrageenan-induced foot pad swelling but exacerbated it. All three proteins inhibit pancreatic phospholipase A2. The phospholipase A2 inhibitory effect of CC10 may be important in regulating the inflammatory responses in the lung.