Overexpression of procollagen gene can cause the extraordinary increase of collagen's synthesis and therefore lead to the keloid and hypertrophic scar. To utilize ribozyme to suppress the expression of procollagen genes, a eukaryotic expression recombinant plasmid containing a dual-ribozyme gene against alpha 1 (I) and alpha 1 (III) procollagen genes was constructed. The ribozyme from in vitro transcription was incubated with target transcripts from recombinant plasmids which separately contained the fragments of the second exons of pro alpha 1 (I) and pro alpha 1 (III) collagen genes under various experimental conditions. The results showed that the dual-ribozyme could efficiently catalyze the specific cleavage of the target RNAs at 37 degrees C, 42 degrees C, 50 degrees C and Mg2+ concentration from 10 mmol/L to 20 mmol/L. This work provided a basis for further study on the ribozyme to suppress the expression of procollagen genes and control the cicatrization.
Base Sequence ; Exons ; Molecular Sequence Data ; Procollagen ; genetics ; RNA ; metabolism ; RNA, Catalytic ; genetics ; metabolism ; Temperature

AIMTo investigate the dynamic expression of hypoxia-inducible factor 1alpha, PHDs and OS-9 in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension.

METHODSSD rats were randomly divided into 5 groups (n = 8) and exposed to hypoxia for 0, 3, 7, 14 or 21 d, respectively. RT-PCR and in situ hybridization were used to determine the expression of mRNA. Immunohistochemistry and Western blot were used to determine the expression of protein.

RESULTSHIF-1alpha protein was poorly positive in control, markedly up-regulated after 3 d and 7 d of hypoxia (P < 0.05, vs group C), and then declined slightly after 14 d and 21 d of hypoxia. HIF-1alpha mRNA increased dramatically after 14 d of hypoxia (P < 0.05, vs group C). PHD1, PHD2 mRNA and protein was positive in group C. PHD2 mRNA and protein were up-regulated after 3 d of hypoxia (P < 0.05, vs group C), reaching its peak after 14 d of hypoxia while PHD1 protein declined after 14 d of hypoxia (P < 0.05, vs group C) without statistic mRNA changing. PHD3 mRNA and protein were detected at low level in control, markedly up-regulated after 3 d of hypoxia (P < 0.05, vs group C), and then PHD3 mRNA kept at high level while PHD3 protein declined after 14 d of hypoxia (P < 0.05, vs 7 d). OS-9 mRNA was positively in control, markedly decreased after 3 d of hypoxia (P < 0.05, vs group C), reaching its lowest lever after 14 d of hypoxia. Linear correlation analysis showed that OS-9 protein was positively correlated with OS-9 mRNA (r = 0.82, P < 0.01) and HIF-1alpha protein (r = 0.57, P < 0.01).

CONCLUSIONHIF-1alpha, PHDs and OS-9 are all involved in the pathogenesis of hypoxic pulmonary hypertension in rats. OS-9 may interact with both HIF-1alpha and PHDs to promote PHD-mediated hydroxylation of HIF-1alpha.

Animals ; Female ; Hypertension, Pulmonary ; metabolism ; physiopathology ; Hypoxia ; complications ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Lectins ; genetics ; metabolism ; Male ; Procollagen-Proline Dioxygenase ; genetics ; metabolism ; Pulmonary Artery ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar

Objective To explore the impact of sinomenine on bleomycin A5-induced pulmonary fibrosis (PF) in rats and the underlying mechanism. Methods MRC-5 cells were cultured and treated with sinomenine to determine its optimal concentration and time through the MTT assay. Subsequently, MRC-5 cells were incubated with 80 μmol/L sinomenine for 48 hours or transfected with miR-21 mimic/a disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS-1) siRNA prior to sinomenine treatment. The expression of miR-21, ADAMTS-1, collagen type 1 (Col1) and collagen type 3 (Col3) was detected by quantitative real-time PCR (qRT-PCR) and/or Western blot analysis. Thirty SD rats were randomly divided into control group, sinomenine group and sinomenine combined with miR-21 agomir group, with 10 animals in each group. Bleomycin A5 were intratracheally administered to establish the PF model. Then, rats in control group, sinomenine group and sinomenine +miR-21 agomir group were treated with 9 g/L sodium chloride solution, sinomenine and sinomenine+miR-21 agomir, respectively. On day 28, all rats were sacrificed. HE and Masson staining was performed in pulmonary tissue. The expression of ADAMTS-1, Col1 and Col3 in pulmonary tissue were detected by qRT-PCR and/or Western blot analysis. ELISA was used to measure serum procollagen type 1 carboxyterminal propeptide (P1CP) and procollagen type 3 aminoterminal propeptide (P3NP) levels. Results Administration of sinomenine decreased miR-21 levels, up-regulated ADAMTS-1 expression, and promoted Col1 and Col3 degradation in MRC-5 cells. Importantly, interfering with the miR-21/ADAMTS-1 signaling pathway partially reversed the promotive effect of sinomenine on Col1 and Col3 degradation. Treatment of SD rats with sinomenine reduced alveolitis and PF scores, decreased serum P1CP and P3NP levels, up-regulated pulmonary ADAMTS-1 expression, and down-regulated Col1 and Col3 expression. However, these effects were reversed by miR-21 agomir. Conclusion Sinomenine promotes Col1 and Col3 degradation and inhibits PF in rats by miR-21/ADAMTS-1 pathway.
Rats ; Animals ; Pulmonary Fibrosis/genetics* ; Procollagen/metabolism* ; Rats, Sprague-Dawley ; Signal Transduction ; Bleomycin/adverse effects* ; Collagen Type III/metabolism* ; MicroRNAs/metabolism*

OBJECTIVETo study the effect of RNA interference (RNAi)-mediated aggrecanase-1 gene silencing on extracellular matrix metabolism of cultured rat costochondral chondrocytes.

METHODSRat costochondral chondrocyte monolayers were obtained by microdissection and digestion. The growth and morphological changes of the chondrocytes were observed after RNAi of aggrecanase-1 gene. The mRNA expression of aggrecanase-1 was detected by RT-PCR method, and aggrecan content was determined by Western blotting.

RESULTSThe specific inhibition of aggrecanase-1 by RNAi produced no adverse effect on the morphology and growth of the chondrocytes. The mRNA of aggrecanase-1 decreased and aggrecan content increased significantly after transfection of the chondrocytes.

CONCLUSIONInhibition of aggrecanase-1 decreases aggrecan degradation in cultured rat chondrocytes. RNAi technique can be a useful means for studying extracellular matrix metabolism in the cartilage.

ADAM Proteins ; genetics ; metabolism ; ADAMTS4 Protein ; Aggrecans ; metabolism ; Animals ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Extracellular Matrix ; metabolism ; Female ; Procollagen N-Endopeptidase ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Humans ; Liver Cirrhosis ; drug therapy ; genetics ; metabolism ; Liver Neoplasms ; drug therapy ; genetics ; metabolism ; Phytotherapy ; Procollagen ; biosynthesis ; genetics ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

Previous studies have demonstrated that enalapril and verapamil seem to attenuate the cyclosporine nephrotoxicity. However, the mechanisms have not been completely understood, especially on molecular events. The aim of this study was to examine the effect of individual or combined treatment on osteopontin, TGF-beta, endothelin-1 and procollagen alpha 1(I) mRNA expressions. Enalapril (50 mg/L in drinking water) and verapamil (0.5 mg/kg/day, subcutaneously), alone or in combination, were administered to rats with chronic cyclosporine nephrotoxicity (cyclosporine, 25 mg/kg/day, subcutaneously) (n = 5 each). Five rats treated with olive oil vehicle were used as control. After 4 weeks, biochemical parameters were measured, and renal cortical mRNA levels were evaluated by Northern blot analysis. Cyclosporine reduced renal creatinine clearance significantly and induced renal cortical osteopontin, TGF-beta, endothelin-1 and procollagen alpha 1(I) gene expressions around 13.5 +/- 1.3, 2.4 +/- 0.2, 1.5 +/- 0.1, 1.9 +/- 0.1 folds, respectively. Individual treatment with enalapril or verapamil significantly suppressed the osteopontin and TGF-beta mRNA expression, but not endothelin-1 and procollagen alpha 1(I). Combined treatment also inhibited the osteopontin and TGF-beta mRNA expression but there was no difference between combined and individual treatment. In conclusion, enalapril or verapamil significantly blunted the cyclosporine-induced osteopontin and TGF-beta gene expressions. However, combined treatment did not show any additive effect.
Angiotensin-Converting Enzyme Inhibitors/therapeutic use* ; Animal ; Calcium Channel Blockers/therapeutic use* ; Cyclosporine/adverse effects ; Drug Therapy, Combination ; Enalapril/therapeutic use* ; Enalapril/administration & dosage ; Endothelin-1/metabolism ; Endothelin-1/genetics ; Gene Expression Regulation/drug effects ; Immunosuppressive Agents/adverse effects ; Kidney Cortex/metabolism ; Male ; Nephritis/drug therapy* ; Nephritis/chemically induced ; Procollagen/metabolism ; Procollagen/genetics ; RNA, Messenger/analysis ; Rats ; Rats, Wistar ; Sialoglycoproteins/metabolism ; Sialoglycoproteins/genetics ; Transforming Growth Factor beta/metabolism ; Transforming Growth Factor beta/genetics ; Verapamil/therapeutic use* ; Verapamil/administration & dosage

Adipocyte differentiation is a very complex process in which whole-cell changes are accompanied. Among them, type I procollagen gene has been shown to specifically decrease during adipocyte differentiation; however, little is known about the molecular mechanism. To examine how type I procollagen gene expression is regulated at the level of transcription during adipocyte differentiation, 3T3-L1 preadipocyte cell line was used as an in vitro model. Northern blot analysis demonstrated that mRNA expression of type I procollagen gene was dramatically reduced during adipocyte differentiation. Time-course analysis indicated that decrease in mRNA expression occurred at early stage of differentiation. Studies on several stable cell lines showed that transcriptional activities of both alpha1 and alpha2 promoters decreased significantly during adipocyte differentiation. Despite extensive deletion-promoter analyses, however, we could not identify the cis-element responsible for the switch-off of type I procollagen gene during adipocyte differentiation, suggesting that the transcriptional repression of this gene occur through general transcription machinery rather than a specific cis-element. In conclusion, down-regulation of type I procollagen mRNA expression during adipocyte differentiation is due to repression of its promoter activity through general transcription machinery.
3T3 Cells ; Adipocytes/cytology/*metabolism ; Animal ; Cell Differentiation/*genetics ; Cell Line ; Collagen Type I/*genetics/metabolism ; Down-Regulation/genetics ; Gene Expression Regulation ; Genes, Reporter ; Kinetics ; Mice ; Mutation ; Procollagen/*genetics/metabolism ; Promoter Regions (Genetics) ; RNA, Messenger/metabolism ; Repressor Proteins/genetics/metabolism ; Transcription, Genetic

Adipocyte differentiation is a very complex process in which whole-cell changes are accompanied. Among them, type I procollagen gene has been shown to specifically decrease during adipocyte differentiation; however, little is known about the molecular mechanism. To examine how type I procollagen gene expression is regulated at the level of transcription during adipocyte differentiation, 3T3-L1 preadipocyte cell line was used as an in vitro model. Northern blot analysis demonstrated that mRNA expression of type I procollagen gene was dramatically reduced during adipocyte differentiation. Time-course analysis indicated that decrease in mRNA expression occurred at early stage of differentiation. Studies on several stable cell lines showed that transcriptional activities of both alpha1 and alpha2 promoters decreased significantly during adipocyte differentiation. Despite extensive deletion-promoter analyses, however, we could not identify the cis-element responsible for the switch-off of type I procollagen gene during adipocyte differentiation, suggesting that the transcriptional repression of this gene occur through general transcription machinery rather than a specific cis-element. In conclusion, down-regulation of type I procollagen mRNA expression during adipocyte differentiation is due to repression of its promoter activity through general transcription machinery.
3T3 Cells ; Adipocytes/cytology/*metabolism ; Animal ; Cell Differentiation/*genetics ; Cell Line ; Collagen Type I/*genetics/metabolism ; Down-Regulation/genetics ; Gene Expression Regulation ; Genes, Reporter ; Kinetics ; Mice ; Mutation ; Procollagen/*genetics/metabolism ; Promoter Regions (Genetics) ; RNA, Messenger/metabolism ; Repressor Proteins/genetics/metabolism ; Transcription, Genetic

Biallelic inactivation of fumarate hydratase(FH) causes type 2 papillary renal cell carcinoma (PRCC2), uterine fibroids, and cutaneous leimyomas, a condition known as hereditary leiomyomatosis and renal cell cancer(HLRCC). The most direct effect of FH inactivation is intracellular fumarate accumulation. A majority of studies on FH inactivation over the past decade have focused on the theory that intracellular fumarate stabilizes hypoxia-inducible factor 1α(HIF1A) through competitive inhibition of HIF prolyl hydroxylases. Recently, a competing theory that intracellular fumarate activates nuclear factor (erythroid-derived 2)-like 2(NRF2) through post-translational modification of its negative regulator. Kelch-like ECH-associated protein 1(KEAP1) has emerged from a computational modeling study and mouse model studies. This review dissects the origin of these two governing theories and highlights the presence of chromatin-structure-regulated targets of transcription factors, which we refer to as "cryptic targets" of transcription factors. One such cryptic target is heme oxygenase I(HMOX1), the expression of which is known to be modulated by the gene product of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4, also known as BRG1).
Animals ; DNA Helicases ; metabolism ; Fumarate Hydratase ; genetics ; metabolism ; Fumarates ; metabolism ; Heme Oxygenase-1 ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Kelch-Like ECH-Associated Protein 1 ; Kidney Neoplasms ; genetics ; metabolism ; Leiomyomatosis ; genetics ; metabolism ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Neoplastic Syndromes, Hereditary ; genetics ; metabolism ; Nuclear Proteins ; metabolism ; Procollagen-Proline Dioxygenase ; metabolism ; Protein Processing, Post-Translational ; Skin Neoplasms ; Transcription Factors ; metabolism ; Uterine Neoplasms

BACKGROUNDLower fluence of 585-nm flashlamp-pumped pulsed dye laser has been successfully used as a nonablative technique in the treatment of wrinkles. The objective of this study was to evaluate the effect of the pulsed dye laser (585 nm) on the production of collagen and the mRNA expression of collagen related gene in fibroblasts in vitro.

METHODSCultured fibroblasts were treated with a 585-nm flashlamp-pumped pulsed dye laser (fluence 3 J/cm(2), 4 J/cm(2), spot size 7 mm, pulse duration 450 micros). The production of collagen and the mRNA expression of transforming growth factor (TGF)-beta1, SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen alpha1, alpha2 in fibroblasts were investigated by colorimetry or real time polymerase chain reaction.

RESULTSThe production of collagen was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm(2) (P < 0.001). The mRNA expression of TGF-beta1, SMAD2, SMAD3, SMAD4, SMAD7 and procollagen I was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm(2) (P < 0.001). No significant difference of mRNA expression of SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen was found between controls and fibroblasts treated with pulsed dye laser with a fluence of 4 J/cm(2) (P > 0.05).

CONCLUSIONSLower fluence (3 J/cm(2)) pulsed dye laser increased the collagen production in fibroblasts by up-regulating TGF-beta1, SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen mRNA expression. These may be the reason it can be effectively used in the treatment of wrinkles.

Analysis of Variance ; Cells, Cultured ; Collagen ; biosynthesis ; Fibroblasts ; cytology ; metabolism ; radiation effects ; Gene Expression ; radiation effects ; Humans ; Lasers ; Procollagen ; genetics ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Smad2 Protein ; genetics ; Smad3 Protein ; genetics ; Smad4 Protein ; genetics ; Smad7 Protein ; genetics ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1