Vol. 3, 549-556, August 1992 Cell Growth & Differentiation 549 Expression of a Preprorelaxin-like Gene during Squamous Differentiation of Rabbit Tracheobronchial Epithelial Cells and Its Suppression by Retinoic Acid’ Anton M. Jetten,2 Susan H. Bernacki, E. Elizabeth Floyd, Nicholas A. Saunders, Jack Pieniazek, and Reuben Lotan Cell Biology Section, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709 [A. M. J., S. H. B., E. E. F., N. A. S.], and Department of Tumor Biology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 [J. P., R. L.] Abstract 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 SQ1O. In this study, we sequenced SQ1O complementary DNA and showed that this gene encodes a preprorelaxin-like protein. The DNA sequence of the coding region of SQ1O has 68% identity with the human preprorelaxin mRNA, whereas the deduced amino acid sequence exhibits 46% identity with human preprorelaxin. An antiserum (peplV-Ab) was raised against a synthetic 22-amino acid oligopeptide of the protein encoded by SQ1O. 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 pro- forms 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 SQ 10. Retinoids which have been shown to inhibit squamous differentiation suppressed the induction of SQ1O protein as well as mRNA in a concentration-dependent manner. The concentration at which retinoic acid caused a 50% inhibition of SQ1O mRNA levels was approximately 5 ni.i. The analogue Ch55 was about 100-fold more effective than retinoic acid and exhibited a 50% effective concentration of about 50 pM. Retinoic acid treatment did not significantly alter the stability of the mRNA encoded by SQ1O. Our study identifies a preprorelaxin-like protein as a new marker for squamous cell differentiation and suggests a role for this hormone in this differentiation process. Introduction The normal tracheobronchial epithelium is a columnar pseudostratified epithelium that is continuously renew- ing itself (1, 2). This epithelium is replaced by a stratified squamous epithelium under a variety of conditions, in- cluding mechanical and toxic injury and vitamin A defi- ciency (2). Squamous differentiation in tracheobronchial epithelial cells shares many biochemical and molecular characteristics with squamous differentiation in other epithelia such as the skin (3, 4). Recently, a multistage model has been proposed for the program of squamous differentiation (2, 4). In the first stage, cells become irreversibly growth arrested. This stage is induced when cultures reach confluence on when logarithmic cultures are treated with phonbol esters on -y-intenfenon (22). In normal cells, irreversible growth arrest appears to be a prerequisite for the second stage of the differentiation program, the induction of the squamous-differentiated phenotype. Changes in many biochemical and molecular markers have been reported to accompany squamous differentiation. These include an increase in the expres- sion of transglutammnase type I (5, 6), cholesterol sulfo- transfenase (7-9), and specific kenatins (10). Squamous differentiation is influenced by several factors, including netinoids, transforming growth factor f,, and phonbol esters (5, 11 , 1 2). Retinoic acid has been shown to act at a very specific stage in this differentiation program. It effectively suppresses the induction of squamous cell markers without blocking irreversible growth arrest (4-10). Previously, we described the isolation of several cDNA3 clones encoding mRNAs that are differentially regulated during squamous cell differentiation of rabbit tracheal epithelial cells (13). In this report, we describe the DNA sequence ofone ofthese cDNA clones, SQ1O. Based on the sequence homology of the coding region of SQ1O, this gene belongs to the nelaxin family. Relaxin is a member of the insulin family and is known to exert its biological effects on various parts of the mammalian reproductive system (14, 15). Jn humans, it is produced by the corpus luteum ofthe ovary during pregnancy (16) and by the prostate (17). This is the first demonstration that preprorelaxin-like molecules are synthesized and Received 2/27/92. 1 This investigation was supported in part by a grant from the David Bruton, Jr. Charitable Trust. The synthetic peptide was produced with support from NIH Cancer Center Core Grant CA 16672. Susan Bernacki was sponsored by a research fellowship ofthe Cystic Fibrosis Foundation. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: cDNA, complementary DNA; RbTE, rabbit tracheal epithelial; kD, kilodalton(s); KLH, keyhole limpet hemocyanin; peplV-Ab, peptide IV antibody; kb, kilobase(s); SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline; RAR, retinoic acid receptor.