Avian SERPINB12 Expression in the Avian Oviduct Is Regulated by Estrogen and Up-Regulated in Epithelial Cell-Derived Ovarian Carcinomas of Laying Hens

Serine protease inhibitors (SERPINs) are involved in a variety of biological processes such as blood clotting, angiogenesis, immune system, and embryogenesis. Although, of these, SERPINB12 is identified as the latest member of clade B in humans, little is known of it in chickens. Thus, in this study, we investigated SERPINB12 expression profiles in various tissues of chickens and focused on effects of steroid hormone regulation of its expression. In the chicken oviduct, SERPINB12 mRNA and protein are abundant in the luminal (LE) and glandular (GE) epithelia of the magnum in response to endogenous or exogenous estrogen. Furthermore, SERPINB12 mRNA and protein increase significantly in GE of cancerous ovaries of laying hens with epithelia-derived ovarian cancer. Collectively, these results indicate that SERPINB12 is a novel estrogen-stimulated gene that is up-regulated by estrogen in epithelial cells of the chicken oviduct and that it is a potential biomarker for early detection of ovarian carcinomas in laying hens and women.     

Human SERPINB12 Is an Abundant Intracellular Serpin Expressed in Most Surface and Glandular Epithelia

The intracellular serine protease inhibitors (serpins) are an important family of proteins that protect cells form proteinase-mediated injury. Understanding the tissue and cellular expression pattern of this protein family can provide important insights into their physiologic roles. For example, high expression in epithelial tissues, such as lung, may suggest a biologic function in cellular defense, secretion, or selective absorption. Although the expression pattern of many of the intracellular serpins has been well described, one member of this class, SERPINB12, has not been carefully examined. We generated a mouse monoclonal antibody directed against human SERPINB12 and delineated its specificity and tissue and cell type distribution pattern through immunoblotting and immunohistochemistry, respectively. This monoclonal antibody was human specific and did not cross-react with other human intracellular serpins or mouse Serpinb12. SERPINB12 was found in nearly all the tissues investigated. In addition, this serpin was found in multiple cell types within individual tissues but primarily the epithelium. These data suggest that SERPINB12, like some other intracellular serpins, may play a vital role in barrier function by providing protection of epithelial cells.     

Chicken egg white cystatin. Molecular cloning, nucleotide sequence, and tissue distribution

A lambda gt11 chicken oviduct cDNA library was screened with a mixed synthetic oligonucleotide corresponding to amino acid residues 81-90 of chicken egg white cystatin, a cysteine proteinase inhibitor. Two initial cDNA clones of 367 and 431 bases were isolated. Both clones contained coding sequences for cystatin from amino acid residue 82 to the carboxyl end plus 3'-untranslated region and a poly(A)+ tail. The two clones utilized different polyadenylation signals located 55 nucleotides apart. Further screening of the library yielded a full-length cystatin cDNA. Sequence analysis indicated that cystatin contains an NH2-terminal extension of 23 amino acids which is probably a signal sequence. The cystatin cDNA hybridized to an mRNA of approximately 0.95 kilobase and was present in varying amounts in all chicken tissues examined. The highest concentration was found in the lung. Gizzard, brain, and heart contained lesser amounts of cystatin mRNA but considerably higher than oviduct. Among a limited number of embryonic tissues examined, significantly higher levels of the mRNA were found in liver and heart tissues when compared with the corresponding adult tissues. These results suggested that the expression of the chicken cystatin gene is tissue-dependent and under developmental control.     

SERPINB11 is a new noninhibitory intracellular serpin. Common single nucleotide polymorphisms in the scaffold impair conformational change

SERPINB11, the last of 13 human clade B serpins to be described, gave rise to seven different isoforms. One cDNA contained a premature termination codon, two contained splice variants, and four contained full-length open reading frames punctuated by eight single nucleotide polymorphisms (SNPs). The SNPs encoded amino acid variants located within the serpin scaffold but not the reactive site loop (RSL). Although the mouse orthologue, Serpinb11, could inhibit trypsin-like peptidases, SERPINB11 showed no inhibitory activity. To determine whether the human RSL targeted a different class of peptidases or the serpin scaffold was unable to support inhibitory activity, we synthesized chimeric human and mouse proteins, in which the RSLs had been swapped. The human RSL served as a trypsin inhibitor when supported by mouse scaffold sequences. Conversely, the mouse RSL on the human scaffold showed no inhibitory activity. These findings suggested that variant residues in the SERPINB11 scaffold impaired serpin function. SDS-PAGE analysis supported this notion as RSL-cleaved SERPINB11 was unable to undergo the stressed-to-relaxed transition typical of inhibitory type serpins. Mutagenesis studies supported this hypothesis, since the reversion of amino acid sequences in helices D and I to those conserved in other clade B serpins partially restored the ability of SERPINB11 to form covalent complexes with trypsin. Taken together, these findings suggested that SERPINB11 SNPs encoded amino acids in the scaffold that impaired RSL mobility, and HapMap data showed that the majority of genomes in different human populations harbored these noninhibitory SERPINB11 alleles. Like several other serpin superfamily members, SERPINB11 has lost inhibitory activity and may have evolved a noninhibitory function.     

SERPINB1 upregulation is associated with in vivo complex formation with neutrophil elastase and cathepsin G in a baboon model of bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) continues to be a major cause of morbidity in premature infants. An imbalance between neutrophil elastase and its inhibitors has been implicated in BPD. Serine protease inhibitor (SERPIN)B1 is an inhibitor of neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (cat G). Recent studies suggest that SERPINB1 could provide protection in the airways by regulating excess protease activity associated with inflammatory lung disorders. In this study, we determined the distribution and ontogeny of SERPINB1 in the baboon lung and characterized the expression of SERPINB1 in baboon models of BPD. SERPINB1 expression was detected in the conducting airway and glandular epithelial cells in addition to neutrophils, macrophages, and mast cells. SERPINB1 mRNA and protein expression increased with advancing gestational age and in the new BPD model. In contrast, SERPINB1 expression levels were decreased in the old BPD model. Furthermore, SERPINB1 was detected as a high-molecular-mass (HMM) complex in lung tissue and bronchoalveolar lavage fluid samples from the BPD group. Analysis of the HMM complex by coimmunoprecipitation showed that these complexes were formed between SERPINB1 and NE or cat G. High-performance liquid chromatography (HPLC) ion trap mass spectrometry verified the presence of SERPINB1 in HMM complexes. Finally, NE activity level was compared between new and old baboon models of BPD and was found to be significantly lower in new BPD. Thus SERPINB1 upregulation in new BPD may be protective by contributing to the regulation of neutrophil proteases NE and cat G.     

Tissue interaction regulates expression of a spasmolytic polypeptide gene in chicken stomach epithelium

The primitive epithelium of embryonic chicken proventriculus (glandular stomach) differentiates, after day 6 of incubation, into luminal epithelium, which faces the lumen and abundantly secretes mucus, and glandular epithelium, which invaginates into mesenchyme and later expresses embryonic chicken pepsinogen (ECPg). So far it is not well understood how undifferentiated epithelial cells differentiate into these two distinct cell populations. Spasmolytic polypeptide (SP) is known to be expressed in surface mucous cells of mammalian stomach. In order to obtain the differentiation marker for proventricular luminal epithelial cells, we cloned a cDNA encoding chicken SP ( cSP ). Sequence analysis indicated that cSP has the duplicated cysteine-rich domain characteristic of SP. Examination of the spatial and temporal expression pattern of cSP gene revealed that, during embryogenesis, cSP was expressed in luminal epithelial cells of the proventriculus, gizzard, small intestine, and lung, but not the esophagus. In the proventriculus, cSP mRNA was first detected on day 8 of incubation and was localized to differentiated luminal epithelial cells. By using cSP as a molecular marker, the effects of mesenchyme on the differentiation of epithelium were analyzed in vitro . On the basis of these data, a model is presented concerning the differentiation of proventricular epithelium.     

Molecular cloning, characterization and mRNA expression analysis of a novel selenoprotein: avian selenoprotein W from chicken

In this study, a novel avian selenoprotein W (SelW) gene was cloned from chicken cerebral tissue. The complete nucleotide sequence of the gene contained a 258 bp open reading frame encoding 85 amino acids. Bioinformatics approaches identified the chicken SelW protein is characterized by a β-α-β-β-β-α secondary structure pattern, wherein β1 and β2 are parallel strands forming a classical β1-α1-β2 motif, which is also observed in thioredoxin-like fold proteins. The protein has a candidate CXXU redox motif which is located in the loop (residues 10-13) between β1 and α1. The 3D structural similarity between mouse and chicken SelW protein suggests that the proteins may exhibit similar functions. Additionally, a selenocysteine insertion sequence (SECIS) element was found in the 3'-untranslated region of the SelW mRNA. The SECIS element was classified as form II. Moreover, we analyzed the mRNA expression of SelW genes in 36 different tissues of 60-day-old chickens; the expression of SelW was detected in all tissues, indicating that SelW is expressed widely in chicken tissue. Hence, we suggest that SelW might play an important role in the biochemical functions of Se in birds.     

转基因鸡生物反应器载体的构建及其表达特性分析

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因,构建了鸡卵清蛋白启动子表达载体和慢病毒载体,以巨细胞病毒 (Cytomegalovirus,CMV)启动子表达载体为对照,转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞,通过荧光和酶活性检测,旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现,鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达,没有表现出明显的细胞特异性,且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上;慢病毒载体感染以上4种细胞后2种标记基因均有表达,在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection,MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明,基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达,而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。     

Sialomucin complex (Muc4) expression in the rat female reproductive tract

Previous studies in our laboratory demonstrated the presence of sialomucin complex (SMC)/Muc4 covering the rat uterine luminal epithelium. SMC/Muc4 expression in the uterus is regulated by estrogen and progesterone and lost at the time of receptivity. In contrast to this hormonal regulation at the uterine luminal surface, SMC/Muc4 in the uterine glandular epithelium, oviduct, cervix, and vagina was constitutively expressed at all stages of the estrous cycle. Furthermore, SMC was expressed in the cervix and vagina of the ovariectomized rat, even though it is not found in the uterine luminal epithelium. Both soluble and membrane-bound forms of SMC were present in these tissues. Immunohistochemical analyses showed distinctive localization patterns of SMC in the various tissues during the estrous cycle. Moreover, the previously unreported expression of SMC/Muc4 in the isthmus, ampulla, and infundibulum of the oviduct suggests potential functions in gamete development. These results indicate that SMC/Muc4 is expressed in most tissues of the female reproductive tract, in which it may have multiple functions. However, hormonal regulation appears to be restricted to the uterine luminal epithelium.     

Structural organization and regulation of the chicken estrogen receptor

We have cloned the chicken estrogen receptor (ER) from a chicken oviduct lambda gt11 library using the human ER cDNA sequence. This chicken ER sequence is virtually identical to the recently published sequence. One noteable difference is an amino acid change from glutamine to arginine located toward the central region of the sequence. The size of the ER protein predicted from the 589 amino acids is approximately 66,000 which fits well with the range of molecular weights previously published for the calf uterine and human ER (65,000-70,000). We observed the size of the chicken ER mRNA to be approximately 7.8 kilobases which is in agreement with the previously published size of 7.5 kilobases. In vivo secondary stimulation of chicken oviduct total RNA with diethylstilbestrol does not induce chicken ER mRNA. A time course following the chicken ER mRNA levels after secondary stimulation with diethylstilbestrol indicated a decrease in mRNA levels 8 h after DES administration. A similar study was performed using progesterone for the secondary stimulation. An increase in the chicken ER mRNA levels was observed 24 h after stimulation with progesterone. Two regions of very high homology were delineated by analyzing the sequence of this chicken ER cDNA and comparing it to the sequences of the human ER, human glucocorticoid, and chicken progesterone receptors and the P75-erbA fusion product of the avian erythroblastosis virus. The first concensus region is 72 amino acids in length and the second region of high homology is 62 amino acids long. Detailed comparisons of these regions for the steroid hormone receptors and v-erb A are presented. Possible functions for the individual regions of high homology are discussed.     

Avidin induction by tissue injury and inflammation in male and female chickens.

1. The occurrence and inducibility of the biotin-binding egg white protein (avidin) in the chicken is not restricted to the oviduct. 2. Inflammatory treatments (intestinal injury, actinomycin D) induced avidin in a number of tissues of young and adult hens and roosters, but not of female rats and mice. Highest avidin concentrations were found in the organs containing epithelial cells and serous membrane. 3. The expression of the avidin gene by tissue injury and inflammation suggests that avidin has a significant function in the injured and inflamed chicken tissues.     

SERPINB12 is a novel member of the human ov-serpin family that is widely expressed and inhibits trypsin-like serine proteinases

Members of the human serpin family regulate a diverse array of serine and cysteine proteinases associated with essential biological processes such as fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation, and apoptosis. Most serpins are secreted and attain physiologic concentrations in the blood and extracellular fluids. However, a subset of the serpin superfamily, the ov-serpins, also resides intracellularly. Using high throughput genomic sequence, we identified a novel member of the human ov-serpin gene family, SERPINB12. The gene mapped to the ov-serpin cluster at 18q21 and resided between SERPINB5 (maspin) and SERPINB13 (headpin). The presence of SERPINB12 in silico was confirmed by cDNA cloning. Expression studies showed that SERPINB12 was expressed in many tissues, including brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary, and intestines. Based on the presence of Arg and Ser at the reactive center of the RSL, SERPINB12 appeared to be an inhibitor of trypsin-like serine proteinases. This hypothesis was confirmed because recombinant SERPINB12 inhibited human trypsin and plasmin but not thrombin, coagulation factor Xa, or urokinase-type plasminogen activator. The second-order rate constants for the inhibitory reactions were 2.5 +/- 1.6 x 10(5) and 1.6 +/- 0.2 x 10(4) M(-1) S(-1), respectively. These data show that SERPINB12 encodes for a new functional member of the human ov-serpin family.     

A retained selection cassette increases reporter gene expression without affecting tissue distribution in SPI3 knockout/GFP knock-in mice

The human serpin, proteinase inhibitor 6 (PI-6/SERPINB6), is a protease inhibitor expressed in many tissues. It inhibits a large number of proteases, including cathepsin G in granulocytes and monocytes. To determine the temporal and spatial distribution of PI-6, mice were generated in which exon 2 of the PI-6 ortholog SPI3 (Serpinb6) was replaced with a green fluorescent protein (GFP) reporter gene. This placed GFP under the control of the regulatory elements and initiation codon of the SPI3 gene. The neomycin selection cassette was flanked by loxP sites to allow excision from the targeted allele. GFP expression in heterozygous and SPI3-deficient mice accurately reflected the tissue distribution of SPI3 in all organs tested and allowed precise comparisons of expression levels. Interestingly, retention of the neomycin cassette in targeted mice resulted in 2-10-fold increases of GFP in leukocytes, but without affecting tissue-specific expression patterns. This is the first example of selection cassette retention specifically increasing reporter gene expression in targeted mice and reinforces the view that selection cassettes must be removed to avoid confounding effects on reporter gene expression patterns.