Expression of a preprorelaxin-like gene during squamous differentiation of rabbit tracheobronchial epithelial cells and its suppression by retinoic acid.

Squamous cell differentiation in tracheobronchial epithelial cells is accompanied by many biochemical and molecular changes. One of the molecular changes in rabbit tracheal epithelial (RbTE) cells is the differential expression of a squamous cell-specific mRNA encoded by the complementary DNA SQ10. In this study, we sequenced SQ10 complementary DNA and showed that this gene encodes a preprorelaxin-like protein. The DNA sequence of the coding region of SQ10 has 68% identity with the human preprorelaxin mRNA, whereas the deduced amino acid sequence exhibits 46% identity with human preprorelaxin. An antiserum (pepIV-Ab) was raised against a synthetic 22-amino acid oligopeptide of the protein encoded by SQ10. Immunoblot analysis of cellular extracts of squamous-differentiated cells showed that this antiserum reacted with proteins of 22 and 20 kilodaltons, possibly constituting prepro- and proforms of this protein. These proteins were undetectable in undifferentiated RbTE cells. In agreement with these observations, PepIV-Ab specifically stained the cytosol of squamous-differentiated RbTE cells but failed to stain undifferentiated cells. PepIV-Ab recognized a 20 and 16 kilodalton polypeptide in medium conditioned by squamous-differentiated RbTE cells, indicating that the prorelaxin-like protein is secreted. The amino acid sequences of three peptides that were obtained after tryptic digestion of the secreted 16 kilodalton protein were identical to sequences encoded by SQ10. Retinoids which have been shown to inhibit squamous differentiation suppressed the induction of SQ10 protein as well as mRNA in a concentration-dependent manner. The concentration at which retinoic acid caused a 50% inhibition of SQ10 mRNA levels was approximately 5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of transglutaminase activity in rabbit tracheal epithelial cells. Regulation by retinoids

Rabbit tracheal epithelial cells undergo terminal cell division, start to express a squamous phenotype, and form cross-linked envelopes when reaching the plateau phase of the growth curve. This terminal differentiation is accompanied by a 20-30-fold increase in the activity of the cross-linking enzyme transglutaminase. This activity is found almost solely in the particulate fraction of homogenized cells and can be solubilized by nonionic detergents. This transglutaminase crossreacts with a monoclonal antibody raised against type I transglutaminase, but does not react with an antiserum against type II transglutaminase. The tracheal transglutaminase contains a protein subunit of approximately 92 kDa. The omission of epidermal growth factor from the medium or the addition of fetal bovine serum, conditions that induce terminal cell division and expression of a squamous phenotype, enhance transglutaminase activity. High calcium concentrations only stimulate transglutaminase activity after the cells become committed to terminal cell division. Retinoids, which inhibit the expression of the squamous phenotype but not terminal cell division, inhibit the enhancement in transglutaminase activity induced by either confluency or serum, indicating that this enzyme activity is under the control of retinoids. Some retinoids are active at concentrations as low as 10(-12) M. The ability of retinoids to inhibit transglutaminase activity correlates well with their capacity to bind to the retinoic acid-binding protein. Our results show that the increase in transglutaminase activity correlates with the induction of the terminal differentiated phenotype and suggest that this enzyme can function as a marker for this program of differentiation of rabbit tracheal epithelial cells in culture. Our results identify the transglutaminase as type I transglutaminase and are in agreement with the concept that this transglutaminase is involved in the formation of cross-linked envelopes.     

The complete amino acid sequence of the human transglutaminase K enzyme deduced from the nucleic acid sequences of cDNA clones.

In order to study the expression and role of transglutaminases in the formation of the cross-linked cell envelope of human epidermis, we have used a synthetic oligonucleotide encoding the consensual active site sequence of known transglutaminase sequences. By Northern blot analysis, newborn foreskin epidermis expresses three different mRNA species of about 3.7, 3.3, and 2.9 kilobases while normal cultured epidermal keratinocytes express only the 3.7- and 2.9-kilobase species. The largest species corresponds to a known ubiquitous tissue type II or transglutaminase C activity, the smallest corresponds to a known type I or transglutaminase K activity, and the mid-sized component apparently encodes a transglutaminase E activity that has recently been shown to be expressed in terminally differentiating epidermis (Kim, H. C., Lewis, M. S., Gorman, J. L., Park, S. C., Girard, J. E., Folk, J. E. & Chung, S. I. (1990) J. Biol. Chem., in press). Using the active site oligonucleotide as a probe, we have isolated and sequenced cDNA clones encoding the transglutaminase K enzyme. The deduced complete protein sequence has 813-amino acid residues of 89.3 kDa, has a pl of 5.7, and is likely to be an essentially globular protein, which are properties expected from the partially purified enzyme. It shares 49-53% sequence homology with the other transglutaminases of known sequence, especially in regions carboxyl-terminal to the active site, and possesses sequences likely to confer its Ca2+ dependence. Interestingly, its larger size is due to extended sequences on its amino and carboxyl termini, absent on the other transglutaminases, that may define its unique properties.     

Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes

This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis.     

Molecular cloning and cDNA structure of the regulatory subunit of type I cAMP-dependent protein kinase from rat brain

Complementary DNA (cDNA) clones encoding the regulatory subunit of the type I cAMP-dependent protein kinase (R-I) were isolated by screening of rat brain cDNA libraries. A 1.5-kilobase (kb) cDNA insert containing the entire coding region was sequenced and full amino acid sequence has been deduced from the nucleotide sequence. The clone encodes for a protein of 380 amino acids that shows 97% homology to the bovine R-I subunit. Northern blot analysis demonstrated two major mRNA species (2.8 and 4.4 kb in size) in rat brain and liver.     

Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.     

Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: differential expression of tau protein mRNAs in human brain.

We have isolated cDNA clones encoding a 383-amino acid isoform of the human microtubule-associated protein tau. It differs from previously determined tau sequences by the presence of an additional repeat of 31 amino acids, giving four, rather than three, tandem repeats in its carboxy-terminal half. The extra repeat is encoded by a separate exon. Probes derived from cDNA clones encoding the three (type I) and four repeat (type II) tau protein isoforms detected mRNAs for both forms in all adult human brain areas examined. However, in foetal brain only type I mRNA was found. Type I and type II mRNAs were present in pyramidal cells in cerebral cortex. In the hippocampal formation, type I mRNA was found in pyramidal and granule cells; type II mRNA was detected in most, though not all, pyramidal cells but not in granule cells. These observations indicate that tau protein mRNAs are expressed in a stage- and cell-specific manner. Tau protein is found in the protease-resistant core of the paired helical filament, the major constituent of the neurofibrillary tangle in Alzheimer's disease. Taken in conjunction with previous findings, the present results indicate that both the three and four repeat-containing tau protein isoforms are present in the core of the paired helical filament.     

Neutrophil attractant/activation protein-1 and monocyte chemoattractant protein-1 in rabbit. cDNA cloning and their expression in spleen cells

Rabbit neutrophil attractant/activation protein-1 (NAP-1) and monocyte chemoattractant protein-1 (MCP-1) were investigated. Rabbit spleen cells stimulated with 5 micrograms/ml of Con A produced both neutrophil and monocyte chemotactic activity. Physicochemical characteristics of those activities obtained by HPLC gel filtration and HPLC chromatofocusing were very similar to those of human NAP-1 and MCP-1, suggesting that rabbit spleen cells produce NAP-1 and MCP-1 after Con A stimulation. A cDNA library was constructed from mRNA purified from Con A-stimulated rabbit spleen cells and screened with oligonucleotide probes. By two rounds of screening, NAP-1 and MCP-1 cDNA were cloned. NAP-1 cDNA comprises 1500 bp with an open reading frame that encodes for a 101-amino acid protein highly similar to human NAP-1. MCP-1 cDNA comprises 607 bp with an open reading frame that encodes for a 124-amino acid protein highly similar to human MCP-1. Expression of NAP-1 and MCP-1 mRNA by rabbit spleen cells was studied. Both Con A- and LPS-stimulated spleen cells expressed NAP-1 and MCP-1 mRNA, but the kinetics of expression were different. Con A rapidly induced high NAP-1 and MCP-1 mRNA expression. LPS also rapidly induced NAP-1 mRNA expression, but high MCP-1 mRNA expression was not observed until 15 h after stimulation. Immunoprecipitation of metabolically labeled NAP-1 and MCP-1 with anti-human NAP-1 or MCP-1 polyclonal antibodies was attempted. Immunoprecipitated rabbit NAP-1 with a molecular mass of about 7 kDa was detected by SDS-PAGE and radioautography, but MCP-1 was not. Cloned rabbit NAP-1 and MCP-1 will give us opportunities to study the role of NAP-1 and MCP-1 in vivo.     

Primary structures of human protein kinase C beta I and beta II differ only in their C-terminal sequences

Two types of cDNA clones encoding human protein kinase C (PKC) were isolated from a spleen cDNA library using rabbit protein kinase C beta I/beta II cDNA as a hybridization probe. Nucleotide sequence analyses of these cDNA inserts revealed complete primary structures of two distinct types of human protein kinase C beta I and beta II which differ only in their C-terminal 50 or 52 amino acid residues. It was concluded that there exist four distinct types of PKC, PKC alpha, beta I, beta II and gamma, in human as well as rabbit, and that the corresponding sequences are strictly conserved among mammalian species.     

Tracheal occlusion stimulates cell cycle progression and type I cell differentiation in lungs of fetal rats

Fetal tracheal occlusion (TO) has been reported to stimulate lung growth but decreases number and maturation of type II cells, effects that vary with gestational age and duration of TO. We examined effects of a novel method of TO (unipolar microcautery to seal the trachea) produced at 19.5-20 days (d) of gestation in fetal rats; fetuses were delivered at term, 22 d. Controls were sham operated and unoperated littermates. TO increased wet lung weight but not dry lung weight or lung DNA and protein. To evaluate further the effects of TO, we examined the cell cycle regulators, cyclins D1 and A, in fetal lungs. Cyclin D1 increased with TO (P < 0.005). TO also increased expression of the type I epithelial cell marker RTI40 (mRNA and protein). TO decreased mRNA for surfactant proteins (SP)-A and -C but did not affect protein levels of SP-A and -B and of RTII70, a type II epithelial cell marker. We conclude that TO by microcautery, even of short duration, has diverse pulmonary effects including stimulating increased levels of cyclin D1 with probable cell cycle progression, type I cell differentiation, and possibly inhibiting type II cell function.     

Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGFβ

In this paper we examined the effects of transforming growth factor β (TGFβ) on the proliferation and differentiation of rabbit tracheal epithelial cells in primary culture. Treatment of these cells with TGFβ inhibits cell proliferation in a time- and dose-dependent manner; concentrations as low as 1 pM are able to inhibit cell growth. Concomitantly, TGFβ causes cells to accumulate in the G0/G1 phase of the cell cycle and a sharp reduction in the ability of the cells to form colonies after subculture at clonal density. These results indicate that TGFβ induces terminal cell division in these cells. The inhibition of cell growth is accompanied by changes in cell morphology and a stimulation of the formation of cross-linked envelopes. TGFβ enhances the levels of transglutaminase activity and cholesterol sulfate, two markers of squamous differentiation. Our results indicate that TGFβ induces terminal squamous cell differentiation in rabbit tracheal epithelial cells. Retinoic acid (RA) does not affect the commitment to terminal cell division induced by TGFβ, but inhibits the expression of the squamous phenotype. Growth of normal human bronchial epithelial cells was affected by TGFβ in a way similar to that of rabbit tracheal epithelial cells. Several carcinoma cell lines tested were quite resistant to TGFβ, whereas growth of one carcinoma cell line was stimulated by TGFβ. These results indicate that a modified response to TGFβ could be one mechanism involved in the aberrant growth control of malignant cells.     

Cytokeratin expression in simple epithelia

To study the regulation of the expression of cytokeratins characteristic of simple epithelia, i.e., human cytokeratins nos. 7, 8, 18, and 19, we prepared several cDNA clones coding for these proteins and their bovine counterparts. In the present study, we describe a cDNA clone of the mRNA coding for human cytokeratin no. 18, which was isolated from an expression library using the monoclonal antibody, KG 8.13. This clone (756 nucleotides, excluding the polyA portion), encodes approximately one-half of the mRNA (approximately 1.4 kb), identifies one mRNA band in Northern-hybridization blots, and specifically selects one mRNA species coding for cytokeratin no. 18, as demonstrated by translation in vitro. Comparison of the deduced amino acid sequence--confirmed by direct amino-acid-sequence analyses of some polypeptide fragments produced by cleavage with cyanogen bromide--indicated that cytokeratin no. 18 is a member of the acidic (type I) subfamily of cytokeratins. It has only limited sequence homologies in common with other intermediate-sized filament proteins, and these are essentially restricted to certain domains of the alpha-helical rod portion. The carboxyterminal tail sequence does not contain glycine-rich elements, thus distinguishing this cytokeratin from those acidic (type I) cytokeratins that are characterized by this feature. The similarities and differences between cytokeratin no. 18 and previously described epidermal cytokeratins are discussed in relation to the differences in the stability of the complexes which this cytokeratin forms with basic (type II) cytokeratins, as well as in relation to possible functional differences of cytokeratins in simple and stratified epithelia.     

Human fibronectin: molecular cloning evidence for two mRNA species differing by an internal segment coding for a structural domain.

Two different fibronectin (FN) mRNA species were detected in the human cell line Hs578T. One species, mRNA I, contains an additional 270 nucleotide long insert (ED) that encodes exactly one of the internally repeated structural domains of the protein. The 90 amino acid extra domain belongs to the so-called type III homology and it is located in the carboxy-terminal half of FN, in between the cell attachment and the heparin binding sites of the protein. The evidence of two mRNAs is provided by the isolation and characterisation of four independent cDNA clones from a library prepared with a synthetic oligonucleotide primer, and it was confirmed by S1 nuclease analysis of cDNA/mRNA hybrids. This kind of analysis also showed that in the human cell line, mRNA I is present at a lower level than mRNA II (the mRNA species without the ED), whilst in human liver, mRNA I is virtually undetectable. Since liver tissue has recently been reported to be the source of plasma FN, our results indicate that the presence of the ED insert could be a particular feature of cellular FN.