Noncollagen Protein Synthesis Research Articles

Salicylate-induced inhibition of collagen and mucopolysaccharide biosynthesis by a chick embryo cell-free system.

Abstract The ability of a chick embryo cell-free system to synthesize collagen, mucopolysaccharide and non-collagen protein in the presence of sodium salicylate was studied. Added creatine phosphate together with endogenous creatine kinase was the ATP generating system. The incorporation of labelled proline or labelled glucose into collagen or mucopolysaccharide respectively depended on ATP level in the cell-free system used. Salicylate inhibited collagen and mucopolysaccharide synthesis to a greater extent than non-collagen protein synthesis. The ability of the cell-free system to hydroxylate labelled protocollagen was inhibited 50% by storage at −18°. Ferrous iron reversed this inhibition. Salicylate prevented the restoration of the enzyme activity by ferrous iron. Incorporation of radioactivity into hyaluronic acid, when labelled UDP-glucuronic acid and UDP-N-acetylglucosamine were supplied, was inhibited 17% by salicylate. Under the same conditions 47% inhibition of incorporation into chondroitin sulphate was seen. This suggests that UDP-N-acetyl-glucosamine-UDP-N-acetylgalactosamine epimerase is inhibited by salicylate.

Enhancement of Procollagen Biosynthesis by p180 through Augmented Ribosome Association on the Endoplasmic Reticulum in Response to Stimulated Secretion

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

Effect of high intensity aerobic exercise and mesterolone on remodeling of Achilles tendon of C57BL/6 transgenic mice

The effect of mesterolone and intensive treadmill training (6 weeks, 5 days/week, means: 15.82 m/min and 45.8 min/day) in Achilles tendon remodeling was evaluated. Sedentary mice treated with mesterolone (Sed-M) or vehicle (Sed-C, placebo/control) and corresponding exercised (Ex-M and Ex-C) were examined. SDS-polyacrylamide gel electrophoresis was used for determining collagen bands and hydroxyproline concentration. Collagen fibril diameter, the area and number of fibrils contained in an area probe, and the ultrastructure of fibroblasts (tenocytes) were determined. The presence of collagen was notable in the tendons of all groups. Collagen alpha(1/)alpha(2) bands in Sed-M, Ex-C, and Ex-M were higher than in Sed-C, as shown by hydroxyproline content, but collagen beta-chain appeared only in Ex-C. Noticeable bands of non-collagenous proteins were found in Sed-M and Ex-M. The number of fibrils in the area probe increased markedly in Sed-M and Ex-C (12-fold), but their diameter and area were unchanged compared with Sed-C. In Ex-M, the fibril number decreased by three-fold to 3.5-fold compared with Sed-M and Ex-C, whereas diameter and area increased. Sed-C tenocytes appeared quiescent, whereas those in the other groups seemed to be engaged in protein synthesis. The density of tenocytes was smaller in Sed-C than in Ex-C, Sed-M, and Ex-M. Thus, mechanical stimuli and mesterolone alter the morphology of tenocytes and the composition of the tendon, probably through fibrillogenesis and/or increased intermolecular cross-links. The ergogenic effect is evidenced by the activation of collagenous and non-collagenous protein synthesis and the increase in the diameter and area of collagen fibrils. This study might be relevant to clinical sports medicine.

Neurogenic control of muscle ribosomal protein synthesis.

In vitro protein synthesis of ribosomes extracted from leg muscles of 20 Sprague-Dawley rats denervated 3 and 14 days (high sciatic transections) and 10 control rats was studied. The concentration/mg protein of total ribosomes extracted from the 14-day denervated muscle showed a significant increase. Distribution of muscle polyribosomes on sucrose density gradients was normal in all cases. Noncollagen protein synthesis of the heavy polyribosomes from both early and late denervated muscles showed a significant decrease. SDS polyacrylamide gels suggested that this decrease affected the synthesis of heavy chains of myosin, while the light chains of myosin, actin and tropomyosin had normal structure and amounts. Collagen synthesis of heavy polyribosomes showed slight to moderate increase in only 50 per cent of the cases. Supplementation of ribosomes extracted from denervated muscles with normal muscle soluble enzymes increased their noncollagen synthesis by 66 per cent. This suggests that the neurogenic control of ribosomal protein synthesis is accomplished by hormonal trophic substances contained in the muscle soluble enzymes.

Metabolic Effects of Avocado/Soy Unsaponifiables on Articular Chondrocytes

Avocado/soy unsaponifiable (ASU) components are reported to have a chondroprotective effect by virtue of anti-inflammatory and proanabolic effects on articular chondrocytes. The identity of the active component(s) remains unknown. In general, sterols, the major component of unsaponifiable plant material have been demonstrated to be anti-inflammatory in vitro and in animal models. These studies were designed to clarify whether the sterol content of ASU preparations were the primary contributors to biological activity in articular chondrocytes. ASU samples were analyzed by high pressure liquid chromatography (HPLC) and GC mass spectrometry. The sterol content was normalized between diverse samples prior to in vitro testing on bovine chondrocytes. Anabolic activity was monitored by uptake of 35-sulfate into proteoglycans and quantitation of labeled hydroxyproline and proline content after incubation with labeled proline. Anti-inflammatory activity was assayed by measuring reduction of interleukin-1 (IL-1)-induced synthesis of PGE2 and metalloproteases and release of label from tissue prelabeled with S-35.All ASU samples exerted a similar time-dependent up-regulation of 35-sulfate uptake in bovine cells reaching a maximum of greater than 100% after 72 h at sterol doses of 1–10 μg/ml. Non-collagenous protein (NCP) and collagen synthesis were similarly up-regulated. All ASU were equally effective in dose dependently inhibiting IL-1-induced MMP-3 activity (23–37%), labeled sulfate release (15–23%) and PGE2 synthesis (45–58%). Up-regulation of glycosaminoglycan and collagen synthesis and reduction of IL-1 effects in cartilage are consistent with chondroprotective activity. The similarity of activity of ASU from diverse sources when tested at equal sterol levels suggests sterols are important for biologic effects in articular chondrocytes.

TGF‐β–induced fibrosis and SMAD signaling: oligo decoys as natural therapeutics for inhibition of tissue fibrosis and scarring

Transforming-growth factor-beta (TGF-beta) is a pleiotrophic growth factor that is synthesized by many cells in the body. This growth factor is chemotactic for fibroblasts, stimulates fibroblast proliferation, and increases the synthesis of a number of extracellular matrix proteins including collagens. The TGF-beta activator protein is a transacting factor, which binds to the TGF-beta element in the distal promoter of the COL1A1 collagen gene and induces transcription of this gene. Although transient TGF-beta 1 activity participates in repair and regeneration of tissues, persistent TGF-beta 1 function affects excessive fibrosis and ultimately scarring of both skin and internal organs. Scarring of internal organ (e.g., liver and lung) results in a loss of function and ultimately death may occur. The central issue of this review is that phosphorothioate double-stranded decoys or other decoys decrease procollagen gene expression, procollagen synthesis, and collagen during fibrogenesis. The rationale is that the decoys containing the TGF-beta element or other gene transcription regulatory CIS-elements bind the transacting proteins preventing the latter from binding to the CIS-element in the 5'-flanking region of the natural gene resulting in transcription inhibition. We will, in part, focus on aspects involved in TGF-beta 1-induced fibrosis that occur during fibrogenesis and the use of the dsTGF-beta element containing oligodeoxynucleotide decoys to control excessive collagen synthesis, and deposition resulting from persistent TGF-beta. In our model of regulation of collagen synthesis, these double-stranded oligo decoys act as promoter competitors, binding to the activator protein either in the cytoplasm or in the nucleus. The significance of the proposed studies is that these novel natural antifibrotics will mimic the effect of glucocorticoids on collagen synthesis during fibrogenesis without the unwanted side effects of these steroids. Based on our previous studies on the molecular mechanisms by which glucocorticoids selectively decrease collagen synthesis, designed phosphorothioate oligodeoxynucleotides resistant to nuclease action will mimic the effects of glucocorticoids at the molecular, cellular, and in vivo levels of collagen synthesis. However, the glucocorticoids significantly inhibit noncollagen protein synthesis. Both the single-stranded and double-stranded oligodeoxynucleotide specifically decrease collagen synthesis without an inhibitory effect on noncollagen protein synthesis. In this review, we will specifically ask if TGF-beta-induced collagen synthesis is inhibited in cell culture and in vivo by using the double-stranded oligodeoxynucleotide decoys, will this inhibit fibrogenesis and ultimately scarring?

Collagen Synthesis in Tenocytes, Ligament Cells and Chondrocytes Exposed to a Combination of Glucosamine HCl and Chondroitin Sulfate

Clinical testing of the nutraceuticals glucosamine (glcN) and chondroitin sulfate (CS) has shown efficacy in providing relief from symptoms in osteoarthritic patients. In vitro and in vivo studies support existence of a synergistic relationship upregulating synthetic activity in chondrocytes. A combination of glcN and CS may also be useful as adjunct therapy in sports-related injuries if similar upregulation of collagen synthesis is elicited in accessory ligament and tendon joint tissue. Collagen and non-collagenous protein (NCP) synthesis in cultures of bovine tenocytes, ligament cells and chondrocytes exposed to glcN + CS were assayed by uptake of radiolabeled proline into collagenase-sensitive material. Assay of radiolabel in hydroxyproline (a specific marker for collagen synthesis) following HPLC isolation confirmed the specificity of the metabolic effect. Synthesis of total collagenase-sensitive material was maximally upregulated at physiologically obtainable doses of glcN + CS. Tissue response followed the sequence ligament cells (+69%) > chondrocytes (+56%) > tenocytes (+22%). Labeled hydroxyproline increased by 132% in ligament cells, 27% in tenocytes and 49% in epitendon cells after a 48 h exposure to 5 μg ml−1 glcN + 4 μg ml−1 CS. Low dose combinations of glcN and CS effectively stimulate in vitro collagen and NCP synthesis by ligament cells, tenocytes and chondrocytes. Hence, therapeutic use following accessory joint tissue trauma may help augment repair processes.

P.089 The application of low-intensity-pulsed ultrasound stimulation to accelerate bony maturation in the alveolar ridge

Mandibular osteoradionecrosis is a serious chronic complication which may follow radiotherapy. Therapeutic ultrasound is a highly effective, inexpensive and readily available means of promoting revascularisation and healing. ‘Long wave’ ultrasound increases penetration depth and, therefore, seems to be more appropriate than traditional high frequency ultrasound. The aim of this study was to compare a new treatment using 45 kHz with the current standard 1 MHz machine. A traditional 1 MHz machine, pulsed 1:4, at intensities of 0.1, 0.4, 0.7 and 1.0 W/cm2(SAPA) was compared with a long wave machine, 45 kHz, at intensities of 5, 15, 30 and 50 mW/cm2(SA). The ultrasound was applied to human gingival fibroblasts and mandibular osteoblasts in vitro. Cell proliferation (DNA synthesis) and collagen and non-collagenous protein synthesis assays were performed, using radiolabelled thymidine and proline, respectively. Controls were sham-insonated and all readings were given as a percentage of controls. Fibroblast proliferation increased by 47% at 0.7 W/cm2 (1 MHz) and by 43% at 50 mW/cm2 (45 kHz), and osteoblast proliferation increased by 52% at 1.0 W/cm2 (1 MHz), and by 35% at 30 mW/cm2 (45 kHz). Fibroblast collagen production increased by 48% at 0.1 W/cm2 (1 MHz), and by 44% at 15 mW/cm2 (45 kHz) and osteoblast collagen production increased by 55% at 0.1 W/cm2 (1 MHz) and by 112% at 30 mW/cm2 (45 kHz). Long wave ultrasound was, therefore, capable of inducing a comparable or even higher enhancement of bone formation compared with traditional ultrasound, which, with its greater penetration, may accelerate the healing effect of ultrasound on osteoradionecrosis. The suggested intensity for 45 kHz ultrasound is 30 mW/cm2.

Strontium ranelate—Data on vertebral and nonvertebral fracture efficacy and safety: Mechanism of action

Strontium ranelate is a novel therapy for the treatment of postmenopausal osteoporosis with actions to reduce bone resorption and increase bone formation. In vitro, strontium ranelate has anabolic and antiresorptive activity, increasing collagen and non-collagen protein synthesis, enhancing pre-osteoblast differentiation, inhibiting osteoclast differentiation, and reducing osteoclast function. In animal models, the increase in bone density is closely correlated with increases in biomechanical bone strength. Histomorphometry demonstrates reduced osteoclast surfaces with increased bone formation. Clinical trials in postmenopausal women have demonstrated 3-year fracture efficacy. Reductions in vertebral fracture were seen in patients with and without prevalent vertebral fracture. Nonvertebral fractures were also significantly reduced. In a subgroup of patients at high risk for hip fracture, there was a significant reduction in hip fracture risk. Strontium ranelate is well tolerated with nausea, diarrhea, headache, and dermatitis more frequent in treated patients only for the first 3 months of therapy. Together, these data suggest that strontium ranelate is a well-tolerated and effective therapy for postmenopausal osteoporosis reducing vertebral and nonvertebral fracture by a novel dual antiresorptive and anabolic action on bone.

Strain-Dependent Control of Transforming Growth Factor-?? Function in Osteoblasts in an In Vitro Model: Biochemical Events Associated with Distraction Osteogenesis

Distraction osteogenesis is an important clinical method for increasing bone mass, but its effects on bone-forming cells are not well understood. In this study, the authors asked how the mechanical forces that occur during this procedure alter specific osteoblast activities such as matrix synthesis, the rate of cell replication, and enzyme activities. The authors further asked whether these changes relate to differences in the biochemical response of osteoblasts to transforming growth factor-beta (TGF-beta), a potent regulator of bone formation. Osteoblasts were plated on flexible, collagen-coated membranes. One group was unstrained, a second group experienced a single maximum strain load once every 6 hours to simulate intermittent force associated with a distraction protocol of four screw turns per day, and a third group was strained continuously for 24 hours. In the third group, some cell cultures were allowed to recover from strain before analysis. Subsequently, each group was treated with vehicle or TGF-beta at 12 pM (0.3 ng/ml) or 120 pM (3 ng/ml). Data were collected from a minimum of 15 replicate cell culture wells obtained from at least three separate primary culture preparations. Results were assessed with statistical software. Differences were considered significant with values of p < 0.05. Both strain protocols increased basal osteoblast DNA synthesis but suppressed the relative stimulatory effect of TGF-beta on this event. However, neither intermittent nor continuous strain significantly altered collagen or noncollagen protein synthesis or the relative effect of TGF-beta on these processes in osteoblasts. Basal alkaline phosphatase activity, an intermediate marker of osteoblast differentiation and an early marker of matrix mineralization, decreased significantly in response to continuous strain or to TGF-beta treatment, and even more so in response to both conditions. In addition, TGF-beta binding to the type III TGF-beta receptor was increased in proportion to strain intensity. This study shows that cyclic strain can alter osteoblast activity in multiple ways and predicts that TGF-beta has different effects during the distraction process on osteoblasts and therefore on their ability to effect bone formation. They further indicate that mechanical load permits early aspects of osteoblast activation but delays in part later biochemical parameters associated with mineralization to allow new bone growth before consolidation.

5-Fluorouracil Selectively Inhibits Collagen Synthesis

Fibroproliferative disorders, such as Dupuytren's contracture of the hand, are characterized by excessive production of collagen. 5-Fluorouracil has been used to treat fibroproliferative disorders of the eye and skin and is thought to inhibit thymidylate synthetase blocking DNA replication. 5-Fluorouracil has been shown to down-regulate fibroblast proliferation and differentiation in vitro. This study investigated the dose-dependent effect of 5-fluorouracil on fibroblast extracellular matrix production. Fibroblasts were derived from tendon and primary Dupuytren's disease of the hand, a fibroproliferative disorder of the palmar aponeurosis (n = 4 patients). Total collagen synthesis was determined by means of the incorporation of radiolabeled proline. Fibroblast secretion of the profibrotic factor transforming growth factor-beta1 (TGF-beta1) was measured by a sandwich enzyme-linked immunosorbent assay. Gene expression of collagen types I and III and TGF-beta1 were quantified by means of reverse-transcriptase polymerase chain reaction assays. The authors found that 5-fluorouracil caused a dose-dependent, selective, and specific decrease in collagen production by Dupuytren's fibroblasts compared with noncollagenous protein synthesis. By contrast, procollagen types I and III mRNA were unaffected by 5-fluorouracil treatment. These changes did not appear to be mediated by alterations in the endogenous secretion of TGF-beta1 or its autocrine effect on collagen metabolism. The clinical implication is that 5-fluorouracil could possibly reduce extracellular matrix production and therefore reduce recurrence of Dupuytren's disease of the hand.

Strontium ranelate: A new treatment for postmenopausal osteoporosis with a dual mode of action

In vitro, strontium ranelate increases collagen and noncollagen protein synthesis by mature osteoblast-enriched cells. Its effects on bone formation were confirmed as the drug enhanced preosteoblastic cell replication. In the isolated osteoclast, preincubation of bone slices with strontium ranelate-induced dose-dependent inhibition of the bone-resorbing activity of treated rat osteoclast. Strontium ranelate dose-dependently inhibited preosteoclast differentiation. Its effect in postmenopausal women with established osteoporosis was assessed during an international, prospective, double-blind, randomized, placebo-controlled phase 3 program comparing strontium ranelate 2 g daily with placebo. The 3-year analysis of the phase 3 study, Spinal Osteoporosis Therapeutic Intervention, evaluating the effect of strontium ranelate 2 g/day on vertebral fracture rates, revealed a significant 41% reduction in the relative risk of patients experiencing new vertebral fracture with strontium ranelate over 3 years. A second phase 3 study showed a significant reduction in the relative risk of experiencing a nonvertebral fracture in the group treated with strontium ranelate over 3 years. These results show that strontium ranelate is a new, effective, and safe treatment for vertebral and hip osteoporosis, with a unique mode of action, increasing bone formation and decreasing bone resorption leading to a rebalance of bone turnover in favor of bone formation.

Changes of the Rat Dentin Matrix Proteins Affected by Long-term Administration of Hydroxyethylidene-1,1-bisphosphonate (HEBP).

Bisphosphonates are widely known as inhibitors of formation and resorption of mineralized tissues. However, it is uncertain how bisphosphonates affect calcium phosphate precipitation and matrix synthesis in mineralized tissues. Several histological approaches have been done to study hard tissues affected by bisphosphonates. In this study, we used a bisphosphonate for biochemical investigation to study the mechanisms of biological mineralization and considered the effects of the bisphosphonates.Hydroxyethylidene-1, 1-bisphosphonate (HEBP) was administered to rats by subcutaneous injection of 10mg P/kg for seven weeks. The incisors of the rats were removed and the dentin matrix proteins were analyzed biochemically. The amount of matrix proteins was relatively increased in the incisors of the experimental rats, though mineralization of the incisors of the those rats was reduced compared with that of the control rats. Dentin phosphophoryns, unique phosphoproteins of dentin, were also elevated in the experimental rats. Nevertheless, the composition of other non-collagenous proteins of dentin was essentially unchanged by the treatment.Inhibition of mineralization by bisphosphonate may not be mediated by inhibition Of synthesis of noncollagenous matrix proteins, but mainly by inhibition of calcium phosphate deposition. Moreover, it is possible that the synthesis of phosphophoryns was promoted by the treatment.

Effect of Orthovanadate on Collagen and Fibronectin Synthesis, and Alkaline Phosphatase Activity in MC3E3-E1 Osteoblast cells

Purpose : The purpose:of this study was to know the effect of inhibition of protein dephosphorylation on the synthesis of collagen and fibronectin (FN), alkaline phosphatase (ALP) activity, and the formation of bone nodule in MC3T3-E1 osteoblasts using orthovanadate (OVA) which is a potent protein tyrosine phosphatase (PTPases) inhibitor. Materilas and Methods : The synthesis of collagen, noncollagenous protein (NCP), and percent collagen in MC3T3-E1osteoblasts with or without OVA treatment according to concentration and time sequence was determined by incorporation of []-proline, synthesis of FN by [] methionine incorproated immunoprecipitation after treatment with 100 OVA for 24 hours, mRNA expression of collagen and FN by Northern blotting, activity of ALP by spectrophotometric method, and formation of bone nodule by staining method. Results : OVA increased collagen and NCP synthesis concentration dependently, until 12 hours in short-time culture, and time dependently through the differentiation until 29 days, however, there was no significant effect on the percent collagen production. OVA increased percent collagen synthesis significantly at 6 hours, and decreased in a long time culture. Total FN synthesis and FN synthesis in cell layer were increased by OVA, however, FN synthesis in medium was not changed. OVA decreased collagen mRNA level dose-dependently and increased the steady-state level of FN mRNA. OVA inhibited activity of ALP in both short and long-time culture. OVA inhibited bone nodule formation in MC3T3-E1 osteoblasts. Conclusion : These results indicate that the inhibition of PTPase by OVA increased the synthesis of collagen, FN, and decreased ALP activity and it resulted in the inhibition of bone formation in MC3T3-E1 osteoblast cells.

Effects of combined administration of transforming growth factor-β1 and epidermal growth factor on properties of the in situ frozen anterior cruciate ligament in rabbits

The mechanical properties of tendon autografts used in reconstruction of the anterior cruciate ligament (ACL) are reduced after surgery. Previous studies showed that growth factors such as transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF) can stimulate fibroblast proliferation and increase collagen and noncollagenous protein synthesis by these cells. These factors might be useful, therefore, in preventing graft deterioration after transplantation or accelerating mechanical restoration of the deteriorated graft. The purpose of our study, therefore, was to clarify the effects of TGF-β1 and EGF on biomechanical properties using an in situ freeze–thaw ACL model in the rabbit. A total of 142 rabbits underwent the freeze–thaw treatment in the right ACL and were then divided into four groups. Group I served as a freeze–thaw, but otherwise untreated control. In Group II, a delivery vehicle (fibrin sealant) alone was applied. In Group III, 4-ng TGF-β1 and 100-ng EGF mixed with the vehicle were applied. In Group IV, higher doses (2-μg TGF-β1 and 50-μg EGF) of growth factors were mixed with the vehicle. The groups were compared at 6 and 12 weeks on the basis of mechanical properties, water content, and histological and ultrastructural observations. The cross-sectional area of Group III (average, 7.1 mm 2) was significantly less than that of Groups I, II, and IV (9.0, 8.2, and 9.4 mm 2, respectively) at 12 weeks. The tensile strength of Group III (62.2 MPa) was significantly greater than that of Groups I, II, and IV (35.6, 43.7, and 36.9 MPa, respectively) at 12 weeks, while the water content of Group III (70.7%) was significantly lower than that of Group I (75.2%). No other significant differences occurred among Groups I, II, and IV. A unimodal distribution of collagen fibril diameters was noted in Groups I and II, while a bimodal pattern was found in Group III. This study demonstrated that low-dose application of TGF-β1 and EGF significantly inhibited not only the increased water content and cross-sectional area, but also the decreased tensile strength caused by the freeze–thaw treatment, while a high dose of TGF-β1 and EGF does not have the same beneficial effects.

Secondary Causes of Osteoporosis

Secondary Causes of Osteoporosis

Rational basis for oligodeoxynucleotides to inhibit collagen synthesis in lung fibroblasts and primary fibroblasts from liver granulomas of Schistosoma mansoni-infected mice

Hepatocellular carcinoma is associated with liver fibrosis. Murine schistosomiasis infection offers a model to study hepatic fibrogenesis. Single-stranded phosphorothiate oligodeoxynucleotides containing the TGF-β regulatory element have been shown to regulate the transcription of this gene and effectively inhibit collagen synthesis in primary fibroblasts isolated from schistosomiasis-induced hepatic granulomas. While the single-stranded oligos did not decrease collagen and non-collagen protein synthesis below control levels, their double-stranded modified and unmodified counterparts did. Competitive cold oligodeoxynucleotide gel mobility shift analysis using control fibroblast nuclear extract demonstrated that the single-stranded oligos diminished binding of the TGF-β activator protein to the TGF-β regulatory element while the double-stranded oligos totally inhibited this binding. TGF-β element containing single-stranded phosphorothioate oligodeoxynucleotides and their double-stranded counterparts may be successful therapeutic agents to inhibit hepatic fibrogenesis and associated hepatocellular carcinoma.

Relationship between glucocorticoid-mediated early decrease of protein synthesis and the steady state decreases of glucocorticoid receptor and TGF-β activator protein

The present studies were undertaken to better elucidate the mechanism(s) by which glucocorticoids inhibit the process of tissue repair. The aim was to determine the importance of the effect of glucocorticoids on decreasing the nuclear TGF-β activator protein. The relationship amongst inhibition of noncollagen protein synthesis and the steady state levels of glucocorticoid receptor and the TGF-β activator protein was examined. Both collagen and noncollagen synthesis were determined in skin fibroblast cell culture and in dermis. Fetal rat skin fibroblasts were treated for 24 h with dexamethsone. Noncollagen protein synthesis was decreased to approximately one-half that of collagen synthesis. Similar results were obtained in dermis in vivo. At 48 h, dexamethasone treatment resulted in practically no nuclear glucocorticoid receptor being noted and a 40–45% steady state decrease of the TGF-β activator protein. We have recently reported that the TGF-β activator protein exists as a protein complex with SP1 and NFKB (p 49). The present data indicate that although the marked decrease of the nuclear glucocorticoid receptor DNA binding following dexamethasone treatment is not comparable to the early 24 h decrease of noncollagen protein synthesis, the decrease of the TGF-β activator protein complex binding to DNA is. The present studies indicate the importance of the effect of dexamethasone on the steady state level of the TGF-β activator protein complex in the glucocorticoid-mediated process inhibition of tissue repair and the relationship of this decrease to the earlier inhibition of protein synthesis.

Stabilization of RNA Strands in Protein Synthesis by Type I Procollagen C-Proteinase Enhancer Protein, a Potential RNA-Binding Protein, in Hepatic Stellate Cells

Type I procollagen C-proteinase enhancer (PCPE) exists in hepatic stellate cells (HSCs) which can produce collagen. The deduced amino acid sequence of PCPE contains motifs specific for RNA-binding proteins. The effect of PCPE on the syntheses of collagen and noncollagenous protein was studied using an HSC clone derived from cirrhotic rat liver. When the cells were cultured in the presence of an antisense oligonucleotide (AS) against PCPE mRNA, the synthesis of noncollagenous protein as well as collagen was reduced compared to the cells cultured with addition of a nonsense oligonucleotide (NS). The extent of the reduction was similar in both syntheses. The total RNA content of the AS-treated cells and NS-treated cells did not differ. In the presence of actinomycin D, however, such total RNA content was decreased more rapidly in the AS-treated cells than in the NS-treated cells. PCPE may be involved in stabilization of RNA strands in noncollagenous protein synthesis as well as collagen synthesis.

Drug-induced acute interstitial nephritis

Drug-induced acute interstitial nephritis