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.     

Annexin I is a stress protein induced by heat, oxidative stress and a sulfhydryl-reactive agent.

Annexin I (also called lipocortin 1) is a 37-kDa member of the annexin family of proteins. It has been proposed to be involved in the regulation of cell growth and differentiation, apoptosis, and inflammation. Previously, we have reported that annexin I displays a chaperone-like function (Kim, G.Y., Lee, H.B., Lee, S.O., Rhee, H.J. & Na, D.S. (1997) Biochem. Mol. Biol. Int. 43, 521-528). To determine the possibility that annexin I is a stress protein, we examined whether expression of annexin I and annexin I mRNA increases in response to stresses in A549 and HeLa cells. Treatments of cells with heat, hydrogen peroxide or sodium arsenite resulted in (a) an increase in annexin I and annexin I mRNA and (b) translocation of annexin I from the cytoplasm to the nucleus and perinuclear region. The annexin I gene promoter region, cloned upstream of a reporter gene, was inducible in response to heat, hydrogen peroxide, and sodium arsenite. These results indicate that annexin I serves as a stress protein and annexins may constitute a new class of stress proteins.     

Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the alpha 1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat alpha 1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase alpha subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep alpha 1 subunit, Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn, was changed to that of the rat, Arg-Ser-Ala-Thr-Glu-Glu-Glu-Pro-Pro-Asn-Asp-Asp. When expressed in HeLa cells, this mutated sheep alpha 1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep alpha 1 cDNA containing only two amino acid substitutions. This double mutation was a Gln-111----Arg and Asn-122----Asp change at the amino terminus and carboxyl terminus, respectively, of the H1-H2 extracellular region. The resistant cells, whether transfected with the rat alpha 1 cDNA, the rat/sheep chimera, or the mutant sheep alpha 1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86Rb+ uptake, and Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Structure and expression of B-myc, a new member of the myc gene family.

The myc family of genes contains five functional members. We describe the cloning of a new member of the myc family from rat genomic and cDNA libraries, designated B-myc. A fragment of cloned B-myc was used to map the corresponding rat locus by Southern blotting of DNA prepared from rat X mouse somatic cell hybrids. B-myc mapped to rat chromosome 3. We have previously mapped the c-myc to rat chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature [London] 306:497-498, 1983) and N-myc and L-myc to rat chromosomes 6 and 5, respectively (S. Ingvarsson, C. Asker, Z. Wirschubsky, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Somat. Cell Mol. Genet. 13:335-339, 1987). A partial sequence of B-myc had extensive sequence homology to the c-myc protein-coding region, and the detection of intron homology further indicated that these two genes are closely related. The DNA regions conserved among the myc family members, designated myc boxes, were highly conserved between c-myc and B-myc. A lower degree of homology was detected in other parts of the coding region in c-myc and B-myc not present in N-myc and L-myc. A 1.3-kilobase B-myc-specific mRNA was detected in most rat tissues, with the highest expression in the brain. This resembled the expression pattern of c-myc, although at different relative levels, and was in contrast to the more tissue-specific expression of N-myc and L-myc. B-myc was expressed at uniformly high levels in all fetal tissues and during subsequent postnatal development, in contrast to the stage-specific expression of c-myc.     

Susceptibility to oxidative stress: proteomic analysis of bronchoalveolar lavage from ozone-sensitive and ozone-resistant strains of mice

Previous studies have shown that the pulmonary response to ozone (O(3)) varies greatly among strains of mice, but the factor(s) and the mechanism(s) that are responsible for this differential susceptibility have not yet been clearly identified. The present study explores the molecular bases for this differential O(3) susceptibility by studying the expression of proteins associated to the epithelial lining fluid (ELF) from two strains of mice, C57BL/6J and the C3H/HeJ, respectively described as O(3)-sensitive and O(3)-resistant. The ELF proteins of these two strains were displayed by two-dimensional gel electrophoresis (2-DE) of bronchoalveolar lavage fluids (BALFs) and the protein patterns obtained with BALF samples of both strains were compared. Two major differences were observed between the BALF 2-DE protein maps obtained from C57BL/6J and C3H/HeJ strains. First, two isoforms of the antioxidant protein 2 (AOP2) were detected in a strain-dependent manner: C3J/HeJ possesses only AOP2a (isoelectric point 5.7) and C57BL/6J exhibits only AOP2b (isoelectric point 6.0). Second, the levels of anti-inflammatory and immunosuppressive Clara cell protein-16 (CC16) were 1.3 times higher in the BALF from resistant C3H/HeJ than from sensitive C57BL/6 mice. Moreover, two 6 kDa isoforms of CC16 with isoelectric points of 4.9 (CC16a) and 5.2 (CC16b) are detected in both strains. Interestingly, the C57BL/6J strain had a twice decreased level of the acidic isoform of CC16 compared to C3H/HeJ. Our results suggest that AOP2 and CC16 might participate in the protection of the pulmonary tract to O(3)-induced lung injury. The possible differential contribution of specific protein isoforms in the differential susceptibility to oxidative stress is discussed.