No abstract

Earlier studies have demonstrated angiotensin II (AngII) and aldosterone (ALDO) each augment cultured adult rat cardiac fibroblast (CFb) collagen synthesis. Whether this involves type I collagen, the major structural protein of the myocardium, and represents a transcriptional event, is uncertain. Accordingly, the influence of AngII and ALDO on transcription and synthesis of fibrillar collagen and on collagenolytic activity was examined in cultured CFb maintained in serum-deprived media. Using concentrations for AngII (10(-7) M) or ALDO (10(-9) M), shown to influence collagen turnover in these cells, we found: a) total collagen synthesis was significantly (p < 0.05) increased (5.4 +/- 0.41 and 4.8 +/- 0.37 vs. control 3.1 +/- 0.55); b) type I collagen production (6590 +/- 710 and 6150 +/- 410 vs. control 4700 +/- 490 ng/mL) in the medium were significantly (p < 0.01) increased; c) type I collagen mRNA expression was also significantly (p < 0.01) increased by AngII (2.0 fold) and ALDO (1.8 fold) compared with control; d) AngII, but not ALDO, significantly (p < 0.05) decreased collagenolytic activity (0.5 fold) compared with control. Thus, AngII and ALDO each increase CFb type I collagen synthesis at the level of transcription and protein synthesis and AngII, but not ALDO, alters collagenolytic activity. Such hormonally mediated alterations in CFb collagen turnover may contribute to the adverse accumulation of fibrillar collagen found in the myocardium in various disease states, where circulating AngII and/or ALDO are increased.

The rat alpha1(I) collagen promoter contains a unique polypurine-polypyrimidine sequence between -141 and -200 upstream of the transcription start site. The polypurine sequence from -171 to -200 (C2) is on the coding strand and the adjacent polypurine sequence from -141 to -170 (C1) is on the non-coding strand. Earlier we demonstrated triplex formation with a polypurine 30 nt parallel triplex-forming oligonucleotide (TFO) corresponding to C1 and inhibition of transcriptional activity of the rat alpha1(I) collagen promoter. In the present work we have tested triplex-forming abilities of shorter (18 nt) purine and pyrimidine TFOs in parallel and antiparallel orientation to the C1 purine sequence. Our results show that purine antiparallel TFOs formed triplexes with the highest binding affinities, while pyrimidine oligodeoxyribonucleotides (ODNs) did not show appreciable binding. Phosphorothioate modification of purine TFOs did not significantly reduce binding affinity. We also demonstrate that preformed triplexes are quite stable when precipitated with ethanol and resuspended in water. Further analysis was carried out using two purine phosphorothioate antiparallel TFOs, 158 APS and 164 APS, designed to bind to the promoter region from -141 to -158 and -147 to -164, respectively, which were found to form triplexes even under physiological conditions. DNase I footprinting experiments showed the ability of these TFOs to protect target sequences in the promoter region; both purine sequences (C1 and C2) were protected in the case of 158 APS. Transfection experiments using preformed triplexes with a reporter plasmid containing the collagen promoter sequence showed significant inhibition of transcription when compared with a control phosphorothioate ODN. The effect of 164 APS was greater than that of 158 APS. These results indicate that this triplex strategy could be used in the down-regulation of collagen synthesis in cultured cells and offer the potential to control fibrosis in vivo.

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of ␣1(I) collagen promoter between nucleotides ؊200 and ؊140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between ؊172 and ؊138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of ␣1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.In response to tissue injury or invasion, a healing response is invoked that ultimately leads to an accumulation of fibrillar type I collagen. This is true for many systemic organs and the heart. Such a healing response, when unabated and invoked in the absence of injury, leads to a progressive interstitial fibrosis that proves pathologic. Parenchymal cell function is compromised by a disproportionate concentration of type I collagen, a characteristic feature of interstitial fibrosis in different organs (1-9). Various stages of organ dysfunction are marked by the activation and repression of type I collagen gene, thereby allowing for the design of specific agents to promote the necessary or adaptive phenotype or to repress the onset of pathologic interstitial fibrosis.A wide array of hormones, cytokines, and growth factors have been implicated in the mediation of fibrous tissue formation (10 -20). Many of these factors mediate their action through transcriptional mechanisms. Therefore, the study of transcriptional regulatory elements within the ␣1(I) and ␣2(I) collagen gene promoters and their trans-acting protein factors is of major interest. Effector cells that bring about fibrosis include interstitial fibroblasts and phenotypically transformed fibroblast-like cells termed myofibroblasts (21).Several cis-acting elements in the ␣1(I) and ␣2(I) collagen genes located on both sides of the transcription start site as well as their trans-acting factors have been identified (for recent reviews, see . Very little is known about the factor(s) bi...

Partial amino acid deprivation of Bacillus subtilis, which evokes the stringent response, initiates sporulation not because the highly phosphorylated guanine nucleotides guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) increase but because GTP decreases. This was shown with a mutant (Myc) partially resistant to mycophenolate, an inhibitor of IMP dehydrogenase. Upon amino acid deprivation, the Myc mutant (62032) showed the usual increase in ppGpp and pppGpp but a reduced decrease in GTP, and only few cells sporulated. Extensive sporulation was restored by the addition of mycophenolate or decoyinine, and inhibitor of GMP synthetase, which caused a further decrease in GTP.