Tubular morphogenesis and mesenchymal interactions affect renin expression and secretion in SIMS mouse submandibular cells.

We have previously immortalized a mouse submandibular gland (SMG) ductal epithelial cell line, SIMS, from pubertal male mice transgenic for the SV40 large T antigen under the control of the adenovirus 5 E1A promoter. Here we demonstrate the role of the extracellular environment in directing not only the morphogenetic behavior of the cells, but also their functional differentiation in terms of renin expression and secretion. First, we measured renin activity of polarized SIMS cells. Low levels of renin are secreted from both the apical and the basolateral domains; the mechanism appears to be direct as no renin was found to be transcytosed across the cell. Second, we studied homotypic and heterotypic mesenchymal cell interactions with SIMS cells. We found that epithelial-mesenchymal coculture in collagen I gels results in branching tubular morphogenesis of SIMS cells and that significant amounts of renin are secreted, probably into the lumen, as the precursor form, prorenin. Third, we investigated the effects of the basement membrane on SIMS cell morphology and function and found that this structure alone is sufficient to allow expression and secretion of both prorenin and active renin. Finally, we established that SIMS cells can express androgen-regulated genes in a transient transfection assay. In addition, in Matrigel cultures androgen receptor expression appears to be induced, suggesting that the SIMS cell line will be useful for further studies on the molecular basis of the observed high-level expression of SMG-specific genes in male mice.

Immortalised mouse submandibular epithelial cell lines retain polarised structural and functional properties.

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.

The functional versatility of CREM is determined by its modular structure.

The CREM gene (cAMP-responsive element modulator) generates both activators and repressors of cAMP-induced transcription by alternative splicing. We determined the exon structure of the CREM gene and have identified new isoforms. We show that CREM isoforms with different structural characteristics are generated by the shuffling of exons to produce proteins with various combinations of functional domains. CREM proteins bind efficiently to CREs and here we demonstrate that the various isoforms heterodimerize in vivo with each other and with CREB. The two alternative DNA binding domains of CREM, which are differentially spliced in the various isoforms, show distinct binding efficiencies, while CREM alpha/CREB heterodimers exhibit stronger binding than CREM beta/CREB heterodimers to a consensus CRE in vitro. We identify the protein domains involved in activation function and find that the phosphorylation domain and a single glutamine-rich domain are sufficient for activation. A minimal CREM repressor, containing only the b-Zip motif, efficiently antagonizes cAMP-induced transcription. In addition, phosphorylation may reduce repressor function, as a CREM beta mutant carrying a mutation of the serine phosphoacceptor site (CREM beta 68) represses more efficiently than the wild-type CREM beta.

Alternative usage of initiation codons in mRNA encoding the cAMP-responsive-element modulator generates regulators with opposite functions.

The cAMP-responsive-element modulator (CREM) gene encodes both antagonists (CREM alpha/beta/gamma) and an activator (CREM tau) of cAMP-responsive transcription by alternative splicing. In adult mouse brain a predominant 21-kDa protein, not corresponding to any previously characterized transcript, is detected with specific CREM antibodies. A developmental switch occurs in brain as expression changes at birth from CREM alpha/beta to the 21-kDa protein. We show that the 21-kDa protein corresponds to S-CREM (short CREM), a protein produced by the use of an internal AUG initiation codon in the CREM tau transcript. S-CREM shares with the other CREM proteins the basic DNA-binding and leucine-zipper dimerization domain. S-CREM functions as a transcriptional repressor of cAMP-induced transcription. Thus, two proteins with opposite functions are generated by alternative translation using two AUG codons within the same reading frame.

Transcriptional antagonist cAMP-responsive element modulator (CREM) down-regulates c-fos cAMP-induced expression.

Protooncogene c-fos is induced by activation of adenylate cyclase through the major cAMP-responsive element (CRE) centered at position -60 of the promoter. cAMP induction is followed by a rapid decrease in transcriptional rate, reminiscent of down-regulation after serum stimulation. Fos protein is known to negatively autoregulate serum-induced transcription of c-fos promoter, but whether Fos is responsible for down-regulation of cAMP-induced transcription is unclear. Here we show that Fos is unable to down-regulate CRE-mediated activation. We present evidence that the transcriptional antagonist CRE modulator (CREM) can bind to c-fos CRE and heterodimerize with activator CRE-binding protein, thereby blocking cAMP induction. Furthermore, expression of antisense CREM enhances c-fos basal and cAMP-induced transcription. CREM does not antagonize serum-induced transcription; therefore, we conclude that down-regulation of c-fos is exerted by different effectors, depending upon which signal transduction pathway is activated. We speculate that, by its c-fos down-regulatory function, CREM may act as an antioncogene.

Virulence dependent and independent regulation of the Bordetella pertussis cya operon.

The Bordetella pertussis adenylate cyclase (cya) operon is composed of four open reading frames, cyaA, B, D and E (Glaser et al., 1988, EMBO J., 7, 3997-4004). The cyaA gene encodes a virulence factor, cyclolysin, a bifunctional protein exhibiting both adenylate cyclase and haemolytic activities while the cyaB, D and E gene products are necessary for cyclolysin transport. We show that the cyaA gene is activated by a promoter located 115 bp upstream from the translational start codon and that transcription is only activated in virulent strains. Termination of transcription occurs 3' to the cyaA structural gene, however there appears to be some read-through into the downstream genes, resulting in full length cyaABDE transcripts. We also identify a second start site of transcription 30 bp upstream from the cyaB gene, in the intergenic cyaA--cyaB region. Transcription is activated from this site in both Vir+ and Vir- strains. Thus, the expression of the virulence associated cyclolysin is positively controlled via a trans-acting protein encoded by the bvg locus while the transport genes show a lower level of constitutive expression which is independent of virulence control.

Bacillus licheniformis alpha-amylase gene, amyL, is subject to promoter-independent catabolite repression in Bacillus subtilis.

Expression of the Bacillus licheniformis alpha-amylase gene, amyL, was temporally activated and subject to catabolite repression both in its natural host and when cloned on a 3.55-kilobase fragment in Bacillus subtilis. A subclone from which the promoter region of amyL and sequences upstream from the promoter were deleted had a low level of amylase activity. Expression of the promoterless gene was still subject to repression by glucose when the gene was present either on a multicopy plasmid or integrated into the B. subtilis chromosome. Catabolite repression occurred independently of the amylase promoter and irrespective of the distance of the promoterless amyL gene from the promoter which transcribed it. The transcriptional start sites of amyL activated by its own promoter and by a vector sequence promoter were determined by S1 mapping. alpha-Amylase-specific mRNA levels were measured in repressing and nonrepressing media, and catabolite repression was found to act at the level of transcription.

cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation mutations and catabolite repression resistance mutations on expression.

Nutrient conditions which trigger sporulation also activate expression of the Bacillus licheniformis alpha-amylase gene, amyL. Glucose represses both spore formation and expression of amyL. A fusion was constructed between the B. licheniformis alpha-amylase regulatory and 5' upstream sequences (amyRi) and the Escherichia coli lacZ structural gene to identify sequences involved in mediating temporal activation and catabolite repression of the amyL gene in Bacillus subtilis. amyRi-directed expression in a variety of genetic backgrounds and under different growth conditions was investigated. A 108-base-pair sequence containing an inverted repeat sequence, ribosome-binding site, and 26 codons of the structural gene was sufficient to mediate catabolite repression of amyL. spo0 mutations (spo0A, spo0B, spo0E, and spo0H) had no significant effect on temporal activation of the gene fusion when the recipient strains were grown in nonrepressing medium. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of amyRi-lacZ. In contrast, a spo0H mutation reduced the repressive effect of glucose on amyRi-lacZ expression. The spo0A effect was relieved by an abrB mutation. Initiation of sporulation is not a prerequisite for either temporal activation or derepression of alpha-amylase synthesis. Mutations causing resistance to catabolite repression in B. subtilis GLU-47, SF33, WLN30, and WLN104 also relieved catabolite repression of amyRi-lacZ.