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)

Expression of keratinocyte transglutamine mRNA revealed by in situ hybridization.

Plasma membrane-bound transglutaminase (TGm) catalyzes the formation of cornified envelopes (CE) in terminally differentiating keratinocytes. The recent cloning of cDNA encoding rabbit TGm allows detailed studies of its gene expression and regulation. In the present paper, we describe the localization of TGm mRNA in rabbit tissues, as well as in normal and psoriatic human skin, as assessed by in situ hybridization. Furthermore, we correlate TGm mRNA localization with the distribution of the TGm protein detected by immunohistochemistry with a specific monoclonal antibody. In rabbit epidermis, TGm mRNA was expressed in suprabasal cells. The TGm protein was detected in the upper stratum spinosum and stratum granulosum. In rabbit esophagus, TGm mRNA and protein were already expressed to a high level in the first suprabasal cell layer, and their expression decreased in the more differentiated cells. In normal human skin, a small amount of TGm mRNA, restricted to the stratum granulosum, was found, whereas psoriatic skin samples contained high amounts of TGm mRNA in the suprabasal layers with a decreasing gradient into the rete ridges, i.e., the involutions of the epidermis into the dermal compartment. The TGm protein was absent from the rete ridges and confined to several cell layers expressing high levels of mRNA. There was virtually no difference between uninvolved psoriatic and normal epidermis.

Primary structure of keratinocyte transglutaminase.

The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine gamma-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of approximately 90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unrelated activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage.

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.

Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: down regulation by retinoids.

Squamous differentiation of rabbit tracheal epithelial cells is accompanied by an approximately 50-fold increase in the activity of type I (epidermal) transglutaminase, while the levels of type II (tissue) transglutaminase remain almost undetectable. To identify a cDNA encoding type I transglutaminase, we screened a library of cDNA clones prepared from poly(A)+ RNA isolated from squamous-differentiated rabbit tracheal epithelial cells. Four overlapping clones (represented by clone pTG-7) which span a range of 2.8 kilobases were identified; partial sequencing of pTG-7 indicated that it encodes a transglutaminaselike protein. pTG-7 hybridized to a 3.6-kilobase mRNA which is distinct from that for type II transglutaminase. pTG-7 mRNA levels were low in proliferative cells, increased dramatically in squamous-differentiated cells, and could be further enhanced by growth of the cells in high concentrations (2 mM) of calcium ions. Retinoic acid, which blocks the expression of the squamous phenotype, prevented this increase in pTG-7 mRNA levels. These changes in levels of pTG-7 mRNA parallel the changes in type I transglutaminase activity observed under similar culture conditions. These data indicate that pTG-7 encodes the mRNA for transglutaminase type I and that expression of this mRNA is negatively regulated by retinoic acid.

Molecular cloning of gene sequences regulated during squamous differentiation of tracheal epithelial cells and controlled by retinoic acid.

A cDNA library was constructed from polyadenylated RNA present in squamous differentiated rabbit tracheal epithelial cells. Screening of the cDNA library was aimed at identifying RNAs that were abundant in squamous cells and expressed at low levels in undifferentiated cells. Two different recombinants were obtained containing inserts, 0.86 and 0.77 kilobases (kb) in size, that hybridized to mRNAs 1.0 and 1.25 kb in length. These RNAs were present at approximately 50-fold higher levels in squamous cells than in proliferative or confluent retinoic acid-treated cells. The increase in the levels of the 1.0- and 1.25-kb RNAs correlated closely with the onset of squamous differentiation and was not related to induction of terminal cell division. Treatment of rabbit tracheal epithelial cells with transforming growth factor beta, which induces squamous differentiation in these cells, also resulted in elevated levels of the 1.0- and 1.25-kb RNAs. The increased levels of these RNAs in squamous cells appeared to a large extent to be regulated at a posttranscriptional level. Retinoic acid not only inhibited the increase in the levels of the 1.0- and 1.25-kb RNAs but also reversed the expression of these RNAs in squamous cells. These results suggest that retinoic acid affects, directly or indirectly, molecular events that induce alterations in the posttranscriptional processing of the transcripts corresponding to the 1.0- and 1.25-kb RNAs.

Calmodulin gene expression during sea urchin development: persistence of a prevalent maternal protein.

Calmodulin gene expression during embryogenesis of the sea urchin Strongylocentrotus purpuratus was investigated. Several identical bacteriophages containing a cDNA insert encoding sea urchin calmodulin (CM-1) were identified by screening a lambda gt10 library of S. purpuratus gastrula-stage cDNAs with a chicken calmodulin cDNA sequence. A 1.2-kb cDNA fragment from CM-1 was subcloned into pUC-8 to give plasmid pCAL-8. pCAL-8 contains a single open reading frame encoding 79 amino acids, a termination codon, and 0.9 kb of 3'-untranslated message. This sea urchin amino acid sequence shows 95% homology to amino acid residues 69-148 of the predicted sequence of chicken calmodulin. Northern analysis showed that pCAL-8 hybridizes to a single size (3.2 kb) of mRNA in both embryonic and adult somatic tissues. Genome blots suggested that there is a single calmodulin gene in the S. purpuratus genome. We used pCAL-8 to study calmodulin mRNA accumulation in S. purpuratus embryos. Calmodulin mRNA is present in the unfertilized egg at the level of a typical rare-class mRNA (1000-2000 transcripts) and accumulates approximately 100-fold to levels representing about 1/10th of 1% of the total mRNA in pluteus-stage cells. Synthesis of calmodulin, identified by two-dimensional gel electrophoresis, shows a similar developmental pattern. However, in spite of the very active synthesis of calmodulin during embryogenesis, most of the calmodulin in the pluteus is apparently provided for by an enormous store of calmodulin in the egg, corresponding to about 2% of the mass of total protein.

Isolation and characterization of the orotidine 5'-monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase.

The multifunctional protein uridine 5'-monophosphate (UMP) synthase catalyzes the final two reactions of the de novo biosynthesis of UMP in mammalian cells by the sequential action of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23). This protein is composed of one or two identical subunits; the monomer weighs of 51,500 daltons. UMP synthase from mouse Ehrlich ascites cells can exist as three distinct species as determined by sucrose density gradient centrifugation: a 3.6 S monomer, a 5.1 S dimer, and a 5.6 S conformationally altered dimer. Limited digestion of each of these three species with trypsin produced a 28,500-dalton peptide that was relatively resistant to further proteolysis. The peptide appears to be one of the two enzyme domains of UMP synthase for it retained only OMP decarboxylase activity. Similar results were obtained when UMP synthase was digested with elastase. OMP decarboxylase activity was less stable for the domain than for UMP synthase; the domain can rapidly lose activity upon storage or upon dilution. The size of the mammalian OMP decarboxylase domain is similar to that of yeast OMP decarboxylase. If the polypeptides which are cleaved from UMP synthase by trypsin are derived exclusively from either the amino or the carboxyl end of UMP synthase, then the size of a fragment possessing the orotate phosphoribosyltransferase domain could be as large as 23,000 daltons which is similar in size to the orotate phosphoribosyltransferase of yeast and of Escherichia coli.

Synthesis and biochemical evaluation of inhibitors of estrogen biosynthesis.

The synthesis and biochemical evaluation of various C19-steroidal derivatives as inhibitors of estrogen biosynthesis are described. Steroids with substitutions on the A or B ring were synthesized by Michael addition of various thiol reagents to appropriate dienone intermediates. An in vitro assay employing the microsomal fraction isolated from human term placenta was used to evaluate aromatase inhibitory properties. Agents exhibiting high inhibitory activity were further evaluated in inital velocity studies (low product formation) to determine apparent Ki values. Several 7alpha-substituted androst-4-ene-3,17-diones were effective competitive inhibitors and have apparent Ki values equal to or less than the apparent Km of 0.063 microM for the substrate androstenedione.