Search

Smoking is accountable for more than 80% of chronic obstructive pulmonary disease (COPD, 3rd cause of death worldwide), which is characterized by emphysema, chronic bronchitis associated to an impaired epithelial permeability. Exposure of the lung to cigarette smoke elicits the expression of elastinolytic cathepsin S (CatS), a cysteine protease, which remains active despite an oxidizing environment [1]. Recently, junctional and/or adhesion molecules were pinpointed as putative CatS targets, suggesting that CatS could proteolytically alter epithelial integrity during COPD.

Rat FAD-dependent sulfhydryl oxidase was purified; partial sequencing indicated that it was homologous to human quiescin Q6. A cDNA (GenBank accession no. AF285078) was cloned from rat seminal vesicles, and active recombinant sulfhydryl oxidase was expressed in Chinese hamster ovary epithelial cells. This 2472-nucleotide cDNA has an open reading frame of 1710 base pairs, encoding a protein of 570 amino acids including a 32-amino acid leader sequence and two potential sites for N-glycosylation. One of them is used and the 64,000 M(r) purified protein was transformed to 61,000 by the action of endoglycosidase F. Northern blotting and reverse transcription-polymerase chain reaction analyses showed that there were small amounts of sulfhydryl oxidase in the rat testis, prostate, lung, heart, kidney, spleen, and liver, and that the gene was highly expressed in seminal vesicles and epididymis. Rat sulfhydryl oxidase cDNA corresponds to the human cell growth inhibiting factor cDNA, which could be a differently spliced form of quiescin Q6. Comparing sulfhydryl oxidase sequences with those of human quiescin Q6 and mammalian and Caenorhabditis elegans quiescin Q6-related genes established the existence of a new family of FAD-dependent sulfhydryl oxidase/quiescin Q6-related genes containing protein-disulfide isomerase-type thioredoxin and yeast ERV1 domains.

The members of the VCS (variable coding sequence) multigene family display extensive evolutionary divergence in the protein-coding region. The first described gene (VCSA1) was found to encode a major 0.7-kb mRNA (VCSA1*1T1) coding for a prohormone-like preproprotein, SMR1-VA1, in the submandibular gland (SMG) of Rattus norvegicus. We report here the cloning of four other VCSAI cDNAs corresponding to mRNAs (VCSAI*1T2 to *1T5) expressed in the SMG. VCSAI*1T1 to *1T4 mRNAs share the three exons previously described and differ in their 3' untranslated regions (UTR). Their differences originate from the alternative utilization of four polyadenylation sites. Comparison of the tissue levels of VCSA1*1T1 and VCSAI*1T4 during post-natal development of the male rat SMG suggests that the poly(A) addition sites are both used at each stage. The fifth RNA transcript (VCSA1*1T5) contains only the first two exons. The nucleotide sequence of the cDNA reveals that VCSA1 has an additional exon (exon 4) which is spliced to exon 2 in VCSAI*1T5. In addition to VCSA1*1T1, at least VCSA1*1T4 and VCSA1*1T5 are actively translated in vivo, as revealed by their association to the polysomal fractions. The protein, P2-VA1, coded by VCSA1*1T5 is 68 amino acids in length and it is likely to be a glycosylated secretory protein. The putative mature P2-VA1 protein completely differs from the SMR1-VA1 pro-protein and very likely has a different function. VCSA1*1T1 is accumulated in the male rat SMG 200-1000-fold more than the other transcripts. Run-on experiments reveal that almost all transcription proceeds several hundred bp downstream from the poly(A) site corresponding to VCSA1*1T1. This suggests that the high levels of VCSA1*1T1 transcript are mainly due to post-transcriptional events.

The mouse submandibular gland (SMG) is an excellent model for the study of many important biological phenomena such as hormonal regulation of differentiation, neurotransmitter control of secretion, epithelial transport, exocytosis and endocytosis as well as the regulation of mouse SMG specific gene expression, in particular, NGF, EGF and renin. The postnatal development and sexual dimorphism of the mouse gland permits the isolation of male SMGs of different ages, corresponding to different stages of differentiation, particularly with respect to the cytodifferentiation of ductal cell types. We have immortalized SMG epithelial cell lines using mice transgenic for the large T antigen of SV40 or polyoma viruses. Epithelial clusters from the dissected glands were placed in culture and cell lines were established from the immortalized population. Two cell lines, SIMS and SIMP, which retain structural and functional characteristics, are described here. The cell lines are immortalised but not transformed, as judged by the absence of anchorage independent growth potential and the lack of tumour formation in athymic nude mice. Confocal and electron microscopy examination demonstrate that SIMP and SIMS cells express E-cadherin and ZO-1 and have features of polarised epithelial cells. In addition, they form spherical cysts with a wide lumen when grown in type I collagen gels. When grown on a filter support SIMS cells form a tight monolayer, exhibit vectorial transport function and show exclusive Na+, K(+)-ATPase localisation to the basolateral domain. We determined the cell type restricted expression of cytokeratin markers in the mouse SMG in vivo and we demonstrate that SIMS and SIMP cell lines express duct-specific cytokeratins. Finally, the expression of a set of differentiation markers, including EGF, NGF and renin, was detected by RT-PCR and by indirect immunofluorescence staining in these lines. Thus, these polarised ductal cell lines, as well as having important intrinsic properties, represent well characterised mouse epithelial models which, until now, have not been readily available for cellular studies.

Summary.Total RNA from mouse epididymides was translated in a cell-free system derived from rabbit reticulocyte lysate. The androgen dependence of a highly represented mRNA(s) was detected. This mRNA(s) encoded for a band of Mr26 000 vizualized by denaturing gel electrophoresis (SDS-PAGE). No other mouse sexual tissues (testis, vas deferens and seminal vesicle), liver, kidney or striated muscle presented such a band. Furthermore, this mRNA(s) was restricted to the caput epididymidis. Two-dimensional gel electrophoresis (2D-PAGE) showed that this band of Mr26 000 was composed of 6 basic polypeptides. This translated protein may correspond to a newly synthesized secretory protein of Mr24 000.The concentration of translatable mRNA(s) encoding for the band of Mr26 000 dropped to 31% of the normal level at 3 days after castration and to 7%, 20 days later. Administration of testosterone to 30-day-castrated mice partly reversed these changes: 32% of the control value was reached after 10 days of treatment. Accumulation of this mRNA(s) was also just detectable (7% of the mature level) at 10 days of age. The high levels attained by 20 days of age (60% of the control value) suggest that the greatest increase of accumulation for this mRNA(s) occurs during the prepubertal period.Keywords:mouse; epididymis; castration; ontogeny; mRNA(s)

Summary.During the reproductive period (spring) under the control of testosterone the epididymis of the viviparous lizard secretes a group of major proteins with an approximate Mrof 19 000 named L protein(s). These proteins are recognized by a specific immunoserum and bind to the heads of spermatozoa. During spring, translation in reticulocyte lysate of RNA from secreting epididymis (stage 6) produced 5 immunoprecipitable bands with Mrvalues from 21 500 to 25 000. Such synthesis is undetectable during sexual rest in summer (stage 1). The 5 bands disappear when translation is performed in the presence of dog pancreas microsomes although a new band of Mr19 000 becomes prominent. This suggests that synthesis of L protein involves two steps, i.e. synthesis of precursors (L preproteins) followed by a maturation process. At least 11 translation products (including L-preproteins) are involved in annual variations that follow the differentiation of the epididymal epithelial cells and their androgen dependency was studied by castration and in-vitro stimulation by testosterone. In these conditions, testosterone is able to control accumulation of RNA corresponding to L preproteins and to a translation product of Mr29 000.

Summary.The hormonal requirements for the regulation of Lv132 mRNA coding for two proteins secreted by the principal cells of the lizard epididymis were examined by organotypic culture experiments. Testosterone, R1881 and corticosterone induced accumulation of Lv132 mRNA in explants from lizards castrated immediately after differentiation of the principal cells. The induction by testosterone was inhibited by the addition of cyproterone acetate. Progesterone and oestradiol alone or in presence of testosterone were ineffective. Unlike the induction by testosterone, the effect of corticosterone did not require binding on the androgen receptor as shown by competition binding studies. Corticosterone failed to induce gene expression in organs containing only reserve cells in their epithelium at the onset of the culture. However, corticosterone plus testosterone had a synergistic effect. These data suggest that testosterone promotes the differentiation of principal cells from reserve cells during the culture time and that a primary action of testosterone is necessary to confer corticosterone responsiveness on this tissue. Furthermore, the primary effects of testosterone could be memorized by the tissue because the corticosterone responsiveness persists after castration.Keywords:epididymis; secretory proteins; androgen; corticosteroid; cell differentiation; lizard

Androgen-dependent sexual differences in the granular convoluted tubules of mouse and rat submandibular glands (SMG) have been extensively reported. We studied two major androgen-dependent mRNAs of the rat SMG encoding proteins named SMR1 and SMR2. To determine which cell type in the SMG is responsible for synthesis of these mRNAs, we performed in situ hybridization with digoxigenin-labeled RNA probes coupled with alkaline phosphatase detection. We show that SMR1 and SMR2 mRNAs are synthesized in the acinar cells of the SMG. A clear difference in SMR1 and SMR2 mRNA levels in male and female is demonstrated. During the course of this study we also confirmed the acinar localization of mRNAs encoding the glutamine/glutamic acid-rich proteins (GRP) of rat SMG. Our data are the first clear evidence of androgen-dependent sexual differences in acinar cells of rat submandibular gland.