Differential responses of Bacillus subtilis rRNA promoters to nutritional stress.

The in vivo expression levels of four rRNA promoter pairs (rrnp(1)p(2)) of Bacillus subtilis were determined by employing single-copy lacZ fusions integrated at the amyE locus. The rrnO, rrnJ, rrnD, and rrnB promoters displayed unique growth rate regulation and stringent responses. Both lacZ activity and mRNA levels were highest for rrnO under all growth conditions tested, while rrnJ, rrnB, and rrnD showed decreasing levels of activity. During amino acid starvation induced by serine hydroxamate (SHX), only the strong rrnO and rrnJ promoters demonstrated stringent responses. Under the growth conditions used, the rrn promoters showed responses similar to the responses to carbon source limitation induced by α-methyl glucoside (α-MG). The ratio of P2 to P1 transcripts, determined by primer extension analysis, was high for the strong rrnO and rrnJ promoters, while only P2 transcripts were detected for the weak rrnD and rrnB promoters. Cloned P1 or P2 promoter fragments of rrnO or rrnJ were differentially regulated. In wild-type (relA(+)) and suppressor [relA(S)] strains under the conditions tested, only P2 responded to carbon source limitation by a decrease in RNA synthesis, correlating with an increase in (p)ppGpp levels and a decrease in the GTP concentration. The weak P1 promoter elements remain relaxed in the three genetic backgrounds [relA(+), relA, relA(S)] in the presence of α-MG. During amino acid starvation, P2 was stringently regulated in relA(+) and relA(S) cells, while only rrnJp(1) was also regulated, but to a lesser extent. Both the relA(+) and relA(S) strains showed (p)ppGpp accumulation after α-MG treatment but not after SHX treatment. These data reveal the complex nature of B. subtilis rrn promoter regulation in response to stress, and they suggest that the P2 promoters may play a more prominent role in the stringent response.

Chondroitin sulphate decreases collagen synthesis in normal and scleroderma fibroblasts through a Smad-independent TGF-beta pathway--implication of C-Krox and Sp1.

Despite several investigations, the transcriptional mechanisms which regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. In this study, we determined the effects of both native ichtyan chondroïtin sulphate (CS) and its derived hydrolytic fragments (CSf) on human normal (NF) and scleroderma (SF) fibroblasts. Here, we demonstrate for the first time that CS and CSf exert an inhibitory effect on type I collagen protein synthesis and decrease the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in NF and SF. These glycosaminoglycan molecules repress COL1A1 gene transcription through a -112/-61 bp sequence upstream the start site of transcription and imply hc-Krox and Sp1 transcription factors. In addition, CS and CSf induced a down-regulation of TbetaRI expression. As a conclusion, our findings highlight a possible new role for CS and CSf as anti-fibrotic molecules and could help in elucidating the mechanisms of action by which CS and CSf exert their inhibitory effect on type I collagen synthesis.

Acyl peptide hydrolase, a serine proteinase isolated from conditioned medium of neuroblastoma cells, degrades the amyloid-beta peptide.

Considerable evidence indicates that the amyloid-beta (Abeta) peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Abeta, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Abeta of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl peptide hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Abeta-degrading enzyme, and we show that the degradation of Abeta by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Abeta peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.

Cartilage oligomeric matrix protein is overexpressed by scleroderma dermal fibroblasts.

Cartilage oligomeric matrix protein (COMP) is an extracellular glycoprotein that belongs to the thrombospondin gene family. It is found predominantly in cartilage, tendon, ligament, and bone. Mutations in the COMP gene have been linked to the development of pseudoachondroplasia and multiple epiphysial dysplasia. COMP influences the organization of collagen fibrils by interacting with collagens I, II and IX. Gene expression profiling of cultured skin fibroblasts suggested that COMP mRNA levels were elevated in scleroderma. We therefore examined COMP expression in SSc and normal skin biopsies. Immunohistochemistry confirmed that COMP protein accumulates in SSc but not normal skin, with SSc skin showing striking deposition in the papillary and deeper dermis. Significant staining was also seen in non-lesional skin from patients. Due to its involvement in the development of fibrosis, TGFbeta was examined for a possible role in regulating COMP expression. Cultured SSc fibroblasts demonstrated greater staining for COMP compared to normal controls prior to stimulation, and TGFbeta-1 induced a large increase in mRNA and protein. Murine fibroblasts engineered to overexpress human COMP demonstrated increased levels of fibronectin and collagen in the extracellular matrix. Taken together, these data demonstrate that COMP is overexpressed in SSc skin and cultured fibroblasts possibly due to autocrine TGFbeta stimulation, and COMP overexpression is sufficient to stimulate excess matrix deposition. By interactions with other matrix proteins and cells, COMP may play a role in pathogenic matrix deposition.

c-Krox down-regulates the expression of UDP-glucose dehydrogenase in chondrocytes.

Chondrocyte glycosaminoglycan (GAG) synthesis is regulated by the availability of UDP-glucuronate, the substrate of glucuronosyl transferases which form the GAG chains in proteoglycans and hyaluronan. UDP-glucose dehydrogenase (UDPGD) is therefore a key enzyme in the synthesis of UDP-glucuronate from glucose. However, the mechanisms regulating its expression in chondrocytes are not fully understood. We investigated the effect of c-Krox, a zinc-finger transcription factor previously shown to modulate several matrix genes, on the synthesis of GAG and transcriptional activity of several UDPGD gene promoter constructs, using transient transfection and decoy experiments in rabbit articular chondrocytes (RACs). We show that overexpression of c-Krox inhibits radiosulfate incorporation into neosynthesized GAG and that the effect was mediated by a cis-sequence located between +18 and +39bp of the UDPGD gene. Since that sequence can also bind Sp1/Sp3 factors, it is likely that c-Krox acts in concert with these proteins to modulate the UDPGD gene expression in articular chondrocytes.