Stimulation Of DNA Synthesis Research Articles

IGF-1 receptor signalling determines the mitogenic potency of insulin analogues in human smooth muscle cells and fibroblasts

Mitogenic activity of insulin and insulin analogues and the involvement of the IGF-1 receptor (IGF-1R) is still a controversial issue. We compared levels of the proteins IGF-1R and insulin receptor (InsR) in fibroblasts and smooth muscle cells from healthy donors and assessed the downstream signalling and growth-promoting activity of insulin and insulin analogues. DNA synthesis was monitored in human fibroblasts and coronary artery smooth muscle cells. Using small interfering RNAs, the levels of IGF-1 and InsR were reduced by 95 and 75%, respectively. Enhanced mitogenic potency of insulin and insulin analogues was observed which correlated with increased levels of IGF-1R and/or IRS-1. A reduction in the IGF-1R level significantly blunted stimulation of Akt phosphorylation by IGF-1, AspB10 and glargine by 72, 58 and 40%, respectively. Akt phosphorylation in response to insulin remained unaffected. Silencing of InsR did not significantly alter Akt phosphorylation in response to IGF-1, AspB10 and glargine. IGF-1R knockdown reduced the stimulation of DNA synthesis in response to IGF-1 and glargine to a level identical to that produced by insulin. These data show a prominent role of IGF-1R/Akt signalling in mediating the mitogenic effects of insulin analogues. Regular insulin stimulates DNA synthesis by exclusively activating InsR, whereas insulin analogues mainly signal through IGF-1R. It is suggested that inter-individual differences in the levels of proteins of the IGF-1R system may function as a critical determinant of the mitogenic potency of insulin analogues.

Mechanisms for the effect of arginine-containing dermorphin analogue on proliferative processes in the gastric mucosa of albino rats

Treatment with synthetic arginine-containing dermorphin analogue sedatin (100 mg/kg, 5 intraperitoneal injections) stimulated DNA synthesis in the gastric mucosa and decreased spontaneous and induced chemiluminescence in homogenates of the stomach from albino rats. Non-arginine sedatin analogue in the same dose had little effect on DNA synthesis and free radical oxidation. Fivefold treatment with NO synthase inhibitor L-NAME (9.3 x 10(-5) mol/kg) suppressed DNA synthesis in the gastric mucosa. Sedatin did not modulate DNA synthesis against the background of L-NAME administration.

Effect of dalargin on DNA synthesis in the gastric mucosa of albino rats

3H-thymidine autoradiography and chemiluminescence study demonstrated pronounced effect of dalargin on the state of the gastric mucosa in albino rats. Dalargin stimulated DNA synthesis in the epithelium of the gastric mucosa and increased buffer capacity of its antiradical and antioxidant systems. Dalargin analogue not containing arginine ([D-Ala2]-leu-enkephalin) had little effect on these parameters. NO synthase inhibitor L-NAME abolished the effects of dalargin on DNA synthesis in the gastric mucosa. Our results suggest that the NO system plays an important role in the effect of dalargin on the gastric mucosa.

Folate Stimulation of Wound DNA Synthesis

Whereas nutrition supply is known to be beneficial to wound healing, the role of folate in wound healing is not clear. The experiment consisted of a surgery on day 0 and a tracer infusion on day 5 after surgery. During the surgery a donor wound was created on the back and indwelling catheters were inserted in the carotid artery and jugular vein in rabbits under general anesthesia. A pharyngostomy feeding tube was also inserted in a group receiving enteral feeding after injury (TEN group, n = 6), but not in other two groups receiving rabbit chow after injury and intravenous amino acids with or without folate during tracer infusion (IVAA and IVAA-folate groups, n = 6 each). On day 5 stable isotope tracers were infused in conscious rabbits to determine the fractional synthetic rate (FSR) of wound DNA, and FSR and fractional breakdown rate (FBR) of wound protein. In the IVAA-folate group 5-methyltetrahydrofolate (the active form of folate) was infused at 0.1 mg/h during the tracer infusion. Serum folate concentration in the IVAA-folate group (380 +/- 78 ng/mL) was greater (P < 0.001) than those in the TEN (20 +/- 6 ng/mL) and IVAA (33 +/- 22 ng/mL) groups. DNA FSRs in the IVAA-folate group (5.02 +/- 1.26%/d) and in the TEN group (4.51 +/- 0.98%/d) were greater (P < 0.01-0.05) than that in the IVAA group (2.87 +/- 0.61%/d). In the IVAA and IVAA-folate groups net protein deposition (FSR-FBR) was correlated with chow intake (P < 0.001-0.002). Folate supplementation has a stimulatory effect on wound DNA synthesis, which would be expected to accelerate wound healing. Nutrition intake is essential for tissue repair by a mechanism that increases net protein deposition in the wound.

L-methionine uptake, incorporation and effects on proliferative activity and protein synthesis in bovine claw tissue explants in vitro

SUMMARYL-methionine is a sulphur-containing nutritionally essential amino acid. It has a number of important roles in epidermal and dermal tissues of the integument of animals. Failure of normal function of these tissues in the hoof (claw) is a cause of lameness in cattle. Little is known about quantitative relationships between post-absorptive concentrations of nutrients including sulphur-containing amino acids and uptake and utilization by epidermis and dermis of the bovine claw. These parameters were studied at the tissue level by use of an established in vitro claw explant system using tissue from cattle of beef or dairy origin and L-[35S]-labelled methionine as tracer. The results showed that uptake of L-methionine by freshly prepared solear explants in Dulbecco's Modified Eagle Medium/F-12 Nutrient Mix (DMEM/F12) (1:1) medium containing 1·0 mmol L-methionine/litre was concentrative after 5–8 min, essentially linear for up to 10 min and became curvilinear thereafter. Maximum uptake and steady state conditions were obtained at approximately 30 min. Further measurements were made following 21 h incubation in culture medium. Under conditions of varying concentrations of L-methionine and measurement of uptake after 30 min, the presence of a saturable curve, that obeyed Michaelis–Menten kinetics, was demonstrated. Values of 3·61 mmol/litre and 5·84 mmol/kg intracellular water/30 min were obtained for KM and Vmax, respectively. Uptake was not influenced by L-cysteine and L-cystine concentrations in the culture media.Similar culture and incubation conditions were used in subsequent studies of DNA and protein synthesis. These showed that rates of incorporation of L-methionine into protein fractions and stimulation of DNA synthesis measured by methyl-thymidine incorporation were dependent on L-methionine concentrations in the medium. Maximal rates occurred at approximately 50 μmol/litre, which is in the normal physiological range, and at 1% of maximum uptake capacity. Examination of histological sections by autoradiography showed localization of L-[35S]-labelled methionine in basal and suprabasal epidermal cells with limited retention in dermis. Measurement, by a range of histological, immunohistochemical, electrophoretic, western blotting and autoradiographic techniques, provided further evidence of L-methionine-dependent regulation of proliferation, differentiation and synthesis of proteins under physiological concentrations, by epidermal horn-forming cells.A key role for L-methionine is suggested in the production of horn in bovine claw. The extrapolation of these in vitro data provides guidance for strategies to optimize methionine supply to claw tissues in vivo. Such extrapolation suggests the appropriateness of delivery of systemic concentrations of 50 μmol L-methionine/litre to maximize proliferative and protein depositional activity in solear epidermis and dermis in vivo.

Lysophosphatidic acid stimulates astrocyte proliferation through LPA1

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates nervous system development and functions through multiple types of LPA receptors. Here we explore the role of LPA receptor subtypes in cortical astrocyte functions. Astrocytes cultured under serum-free conditions were found to express the genes of five LPA receptor subtypes, lpa1 to lpa5. When astrocytes were treated with dibutyryl cyclic adenosine monophosphate, a reagent inducing astrocyte differentiation or activation, lpa1 expression levels remained unchanged, but those of other LPA receptor subtypes were relatively reduced. LPA stimulated DNA synthesis in both undifferentiated and differentiated astrocytes, but failed to do so in astrocytes prepared from mice lacking lpa1 gene. LPA also inhibited [3H]-glutamate uptake in both undifferentiated and differentiated astrocytes; and LPA-induced inhibition of glutamate uptake was still observed in lpa1-deficient astrocytes. Taken together, these observations demonstrate that LPA1 mediates LPA-induced stimulation of cell proliferation but not inhibition of glutamate uptake in astrocytes.

Ceramide kinase uses ceramide provided by ceramide transport protein: localization to organelles of eicosanoid synthesis

Ceramide kinase (CERK) is a critical mediator of eicosanoid synthesis, and its product, ceramide-1-phosphate (C1P), is required for the production of prostaglandins in response to several inflammatory agonists. In this study, mass spectrometry analysis disclosed that the main forms of C1P in cells were C(16:0) C1P and C(18:0) C1P, suggesting that CERK uses ceramide transported to the trans-Golgi apparatus by ceramide transport protein (CERT). To this end, downregulation of CERT by RNA interference technology dramatically reduced the levels of newly synthesized C1P (kinase-derived) as well as significantly reduced the total mass levels of C1P in cells. Confocal microscopy, subcellular fractionation, and surface plasmon resonance analysis were used to further localize CERK to the trans-Golgi network, placing the generation of C1P in the proper intracellular location for the recruitment of cytosolic phospholipase A(2)alpha. In conclusion, these results demonstrate that CERK localizes to areas of eicosanoid synthesis and uses a ceramide "pool" transported in an active manner via CERT.

Antibacterial Peptides - A Bright Future or a False Hope

The increasing number of antibiotic resistant bacterial strains presents an emerging world health problem that demands continued effort to develop new antibacterial compounds. Endogenous antibacterial peptides (ABPs) that are constitutively and/or inducibly produced in tissues exposed to external surroundings represent new candidates for devel- opment of such compounds. Most ABPs target bacterial membranes initially by electrostatic interactions between posi- tively charged amino acids and negatively charged molecules present on bacterial walls, followed by compromise of the permeability barrier of bacterial membranes through the formation of pores, leading to rapid cell death and efficient bacte- rial elimination. Other mechanisms, such as the inhibition of bacterial protein and DNA synthesis or receptor-mediated stimulation of host defense mechanisms are also ascribed to these molecules. The activities of ABPs correlate positively with a gradient of hydrophobicity along the peptide backbone, net positive charge at neutral pH, and secondary structure. More than 850 sequences with antibacterial activity have been described, and this number continues to grow with the ad- dition of newly discovered, as well as synthetic, peptides. Many strategies, including increasing net positive charge, in- creasing net hydrophobicity, conjugation of peptides with lipophilic acids, incorporation of carbonate bonds, and synthesis of their hybrids and truncated sequences that omit hemolytic regions, have been proposed to increase efficiency of syn- thetic ABPs. Additionally peptide-mimicking molecules such as cationic steroid antibiotics (CSAs) may be useful alterna- tives to natural ABPs. Current challenges for practical application of ABPs are the high cost of synthesis/isolation, inacti- vation by blood plasma, confinement by anionic polyelectrolytes, and possible unknown toxicity.

Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes

Glucocorticoids paradoxically exert both stimulatory and inhibitory effects on the proliferation of cultured rat hepatocytes. We studied the effects of dexamethasone, a synthetic glucocorticoid, on the proliferation of cultured rat hepatocytes. The timing of growth factor addition modified the action of high-dose dexamethasone (10(-6) M) on DNA synthesis. When we added transforming growth factor-alpha at the time of plating, 10(-6) M dexamethasone weakly stimulated DNA synthesis by 26% relative to cells cultured in dexamethasone-free media. When we delayed growth factor addition until 24-48 h after plating, 10(-6) M dexamethasone inhibited DNA synthesis by 50%. Using immunological methods, we analyzed the expression and signaling patterns of the ErbB kinases in dexamethasone-treated cells. High-dose dexamethasone stabilized the expression of epidermal growth factor receptor (EGFr) and ErbB3, and it suppressed the de novo expression of ErbB2 that occurs during the third and fourth day of culture in 10(-8) M dexamethasone. High-dose dexamethasone by 72 h suppressed basal and EGF-associated phosphorylation of ERK and Akt. The reduction in ERK1/2 phosphorylation correlated with suppression of a culture-dependent increase in Son-of sevenless 1 (Sos1) and ERK1/2 expression. High-dose dexamethasone in hepatocytes stabilized or upregulated several inhibitory effectors of EGFr/ErbB2 and ERK, including receptor-associated late transducer (RALT) and MKP-1, respectively. Thus 10(-6) M dexamethasone exerts a time-dependent and redundant inhibitory effect on EGFr-mediated proliferative signaling in hepatocytes, targeting not only the ErbB proteins but also their various positive and negative effectors.

Immobilization of Bioactive Fibroblast Growth Factor-2 into Cubic Proteinous Microcrystals (Bombyx mori Cypovirus Polyhedra) That Are Insoluble in a Physiological Cellular Environment

The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.

Cyclic Pressure Stimulates DNA Synthesis through the PI3K/Akt Signaling Pathway in Rat Bladder Smooth Muscle Cells

Previous studies demonstrated that the bladder exhibited severe tissue remodeling following spinal cord injury. In such pathological bladders, uninhibited non-voiding contractions subject bladder cells to cyclic oscillations of intravesical pressure. We hypothesize that cyclic pressure is a potential trigger for tissue remodeling in overactive bladder. Using a custom-made setup, rat bladder smooth muscle cells (SMC) in vitro were exposed to cyclic hydrostatic pressure (40 cm H2O) at either 0.1 Hz or 0.02 Hz frequency for up to 24 h. When compared to static control and cells exposed to 0.02-Hz cyclic pressure, SMC exposed to 0.1-Hz cyclic pressure contained significantly (p < 0.05) higher amounts of DNA. We confirmed that the increase in DNA was due to increased cell proliferation, indicated by increased BrdU incorporation, but not due to decreased apoptosis rates in response to cyclic pressure. In addition, significant (p < 0.05) elevation of Akt phosphorylation in SMC following exposure to cyclic pressure and lack of pressure-induced SMC hyperplasia in the presence of PI3K inhibitors, wortmannin and LY294002, indicated the involvement of the PI3K/Akt pathway in the proliferative response of SMC to cyclic pressure. We concluded that chronic exposure to intravesical pressure oscillation may be a potential trigger for bladder tissue remodeling.

Complement 3 is involved in changing the phenotype of human glomerular mesangial cells

Complement activation contributes to tissue injury in various forms of glomerulopathy and is characterized by deposition of complement components, which accelerates the progression of chronic renal damage. We recently reported that complement 3 (C3), a critical component of the complement system, is associated with the synthetic phenotype of vascular smooth muscle cells. It is possible that C3 stimulates mesangial cells to assume the synthetic phenotype to, in turn, induce glomerular injury and sclerosis. We investigated the role of C3 in the growth and phenotype of mesangial cells. Cultured human mesangial cells (HMCs) expressed C3 mRNA and protein, and levels were increased in response to IFN-gamma and TNF-alpha. HMCs also expressed C3a receptor mRNA and protein. Exogenous C3a stimulated DNA synthesis in HMCs in a dose-dependent manner. C3a decreased expression h-caldesmon mRNA, a marker of the contractile phenotype, and increased the expression of osteopontin, matrix Gla, and collagen type1 alpha1 (collagen IV) mRNAs, which are markers of the synthetic phenotype. C3a decreased expression of alpha-smooth muscle actin in HMCs. Small interfering RNA (siRNA) targeting C3 reduced the DNA synthesis and proliferation of HMCs, increased expression of h-caldesmon mRNA, and decreased expression of osteopontin, matrix Gla, and collagen IV mRNAs in HMCs. These results indicate that C3 causes HMCs to convert to the synthetic phenotype and stimulates growth of mesangial cells, suggesting that C3 may play an important role in phenotypic regulation of mesangial cells in renal diseases.

Paracrine stimulation of vascular smooth muscle proliferation by diadenosine polyphosphates released from proximal tubule epithelial cells

The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.

Tesaglitazar, a PPARα/γ Agonist, Induces Interstitial Mesenchymal Cell DNA Synthesis and Fibrosarcomas in Subcutaneous Tissues in Rats

The development of the dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year carcinogenicity study in rats given 0.3, 1, 3, and 10 micromol/kg tesaglitazar is presented with focus on the findings of subcutaneous fibrosarcomas. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis (immunohistochemical detection of BrdU-labeled cells) and expression of PPARgamma (immunohistochemistry and reverse transcription-polymerase chain reaction) in subcutaneous adipose tissues was assessed in rats administered 1 or 10 micromol/kg for 2 weeks or 3 months. Poorly differentiated subcutaneous mesenchymal sarcomas with a predominant spindle cell appearance occurred at the highest dose level of 10 micromol/kg in both sexes, and these tumors were diagnosed as fibrosarcomas. The 10-micromol/kg dose was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased at 1 and 10 micromol/kg after 2 weeks. The increase in DNA synthesis was still significant at the end of the 12-week treatment at 10 mumol/kg, the dose producing fibrosarcoma. However, at 1 micromol/kg, a dose below the no-observed-effect level for fibrosarcoma, the level of DNA synthesis was similar to control levels at 12 weeks. Immunohistochemical analyses showed no detectable PPARgamma protein in the majority of BrdU-labeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARgamma in these cells. The results suggest that the induction of rat fibrosarcoma by tesaglitazar, at exposures 100-fold above the human therapeutic exposure, may involve proliferation of undifferentiated mesenchymal cells in subcutaneous tissues.

On translocation mechanism of ring-shaped helicase along single-stranded DNA

The ring-shaped helicases represent one important group of helicases that can translocate along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA by using the energy derived from NTP binding and hydrolysis. Despite intensive studies, the mechanism by which the ring-shaped helicase translocates along ssDNA and unwinds dsDNA remains undetermined. In order to understand their chemomechanical-coupling mechanism, two models on NTPase activities of the hexamers in the presence of DNA have been studied here. One model is assumed that, of the six nucleotide-binding sites, three are noncatalytic and three are catalytic. The other model is assumed that all the six nucleotide-binding sites are catalytic. In terms of the sequential NTPase activity around the ring and the previous determined crystal structure of bacteriophage T7 helicase it is shown that the obtained mechanical behaviors such as the ssDNA-translocation size and DNA-unwinding size per dTTPase cycle using the former model are in good quantitative agreement with the previous experimental results for T7 helicase. Moreover, the acceleration of DNA unwinding rate with the stimulation of DNA synthesis by DNA polymerase can also be well explained by using the former model. In contrast, the ssDNA-translocation size and DNA-unwinding size per dTTPase cycle obtained by using the latter model are not consistent with the experimental results for T7 helicase. Thus it is preferred that the former model is the appropriate one for the T7 helicase. Furthermore, using the former model some dynamic behaviors such as the rotational speeds of DNA relative to the T7 helicase when translocation along ssDNA and when unwinding dsDNA have been predicted, which are expected to test in order to further verify the model.

Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication

Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase delta, but not polymerase epsilon or alpha, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase delta complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase delta, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase delta, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.

Local injection of insulin‐zinc stimulates DNA synthesis in skin donor site wound

Our group has reported the ability of local insulin-zinc injection to accelerate skin wound healing. This experiment was conducted to investigate the metabolic mechanisms of locally injected insulin-zinc in accelerating wound healing. A partial thickness skin donor site wound was created on the back, and indwelling catheters were placed in the carotid artery and jugular vein in anesthetized rabbits. On day 7 after injury, the wound was either injected with 0.2 U of insulin-zinc, 0.4 microg zinc (the amount of zinc in 0.2 U of insulin-zinc), or not injected; stable isotope tracers were infused for measurement of wound DNA synthetic rate as a reflection of cell proliferation. Wound protein synthetic and breakdown rates were also measured. The local insulin-zinc injection increased wound insulin concentration from below 5 pmol/L to 1,260+/-780 pmol/L with minor changes in blood glucose concentration that did not require exogenous glucose replacement. In the insulin-zinc-injected wound, the total DNA synthetic rate was increased by approximately 50% (p<0.05 vs. control). In the zinc-injected wound, whereas total DNA synthetic rate was increased (p<0.05 vs. control), net protein deposition (synthesis-breakdown) was less (p<0.05) than those in the control and insulin-zinc groups. We conclude that local insulin-zinc injection stimulates wound DNA synthesis, which would be expected to accelerate wound reepithelialization.

Articular chondrocyte aging and endothelin-1

Objective. To compare in cell culture endothelin-1 (ET-1) production, receptor density, and effect on macromolecular synthesis by articular chondrocytes (AC). Methods. AC were isolated from 1-month and 18-month old rats and cultured as monolayers. They were incubated with ET-1 without or with iNOS inhibitors, nitro- l-arginine methyl ester ( l-NAME) or guanylate cyclase inhibitor, LY83583 and then [ 3H]thymidine, 35SO4 and [ 3H]proline incorporations were measured. The density and affinity for 125I-ET-1 of binding sites, and receptor isotypes were determined. The cells were also treated with interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF−α), and then ET-1 productions measured. As well, the cells were challenged with NOC-5 (nitric oxide donor) or ET-1 and then ET-1 and NO respectively were measured. Results. A concentration-dependent stimulation of DNA, PG, collagen and NO synthesis was obtained when cells were incubated with ET-1 for 24-h. Eighteen-month old chondrocytes incorporated per μg DNA more [ 3H]thymidine, 35SO4 and [ 3H]proline but less NO when challenged with ET-1 than the 1-month old cells. However, strong inhibition of this initial stimulation was seen after 48-h. l-NAME and LY83583 enhanced basal-, and ET-1-induced initial stimulation and completely suppressed late (at 48-h to 72-h) ET-1-induced inhibition, suggesting NO was responsible for this inhibitory effect. Eighteen-month old chondrocytes expressed per μg DNA more high affinity receptors of predominantly ET A subtype. They also produced more ET-1 but less NO under basal conditions and more ET-1 when challenged with IL-1β and TNF-α. NOC-5 inhibited the production of ET-1. Conclusions. Eighteen-month old chondrocytes produce more ET-1, possess more ET-1-specific receptors, and increase more DNA, PG and collagen synthesis when challenged during 24-h with ET-1. NO, which suppresses ET-1 production and the production of which is increased by ET-1, seems to account for the late ET-1-induced inhibition of macromolecular synthesis. The possible implication of ET-1 in aging as related to osteoarthritis is discussed.

Insulin-like growth factor-II regulates the 12-lipoxygenase gene expression and promotes cell proliferation in human keratinocytes via the extracellular regulatory kinase and phosphatidylinositol 3-kinase pathways

To study the relationship between insulin-like growth factor-II (IGF-II) and 12-lipoxygenase (12-LOX) that are upregulated in psoriasis, we monitored 12-lipoxygenase expression in the insulin-like growth factor-II treated human keratinocytes and explored the signaling pathways of 12-lipoxygenase expression. Insulin-like growth factor-II induced 12-lipoxygenase mRNA and protein levels in human keratinocytes through two major signal transduction pathways, namely, the extracellular signaling-regulated kinase (ERK)-mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. The IGF-II-induced upregulation of 12-lipoxygenase was attenuated by pretreating the cells with selective inhibitors or by overexpressing dominant-negative MEK. In addition, treatment of HaCaT cells with the 12-lipoxygenase metabolite 12 (S)-hydroxyeicosatetraenoic acid (12(S)-HETE) directly stimulated DNA synthesis and mitogenesis, and injection of insulin-like growth factor-II into the skin of hairless mice induced epidermal hyperplasia. These results suggest that insulin-like growth factor-II is involved in the pathogenesis of psoriasis as a paracrine inducer of 12-lipoxygenase.

Loss of TGF-β dependent growth control during HSC transdifferentiation

Liver injury induces activation of hepatic stellate cells (HSCs) comprising expression of receptors, proliferation, and extracellular matrix synthesis triggered by a network of cytokines provided by damaged hepatocytes, activated Kupffer cells and HSCs. While 6 days after bile duct ligation in rats TGF-β inhibited DNA synthesis in HSCs, it was enhanced after 14 days, indicating a switch from suppression to DNA synthesis stimulation during fibrogenesis. To delineate mechanisms modulating TGF-β function, we analyzed crosstalk with signaling pathways initiated by cytokines in damaged liver. Lipopolysaccharide and tumor necrosis factor-α enhanced proliferation inhibition of TGF-β, whereas interleukin-6, oncostatin M, interleukin-1α, and interleukin-1β did not. Hepatocyte growth factor (HGF) counteracted TGF-β dependent inhibition of DNA synthesis in quiescent HSCs. Since expression of c-met is induced during activation of HSCs and HGF is overrepresented in damaged liver, crosstalk of HGF and TGF-β contributes to loss of TGF-β dependent inhibition of DNA synthesis in HSCs.