Stimulation Of DNA Synthesis Research Articles

Expression and regulation of the insulin-like growth factor axis components in rat liver myofibroblasts.

Apart from hepatic stellate cells (HSC), liver myofibroblasts (MF) represent a second mesenchymal cell population involved in hepatic fibrogenesis. The IGF system including the insulin-like growth factors I and II (IGF-I, -II), their receptors (IGF-I receptor, IGF-IR; IGF-II/mannose 6-phosphate receptor, IGF-II/M6-PR), and six high affinity IGF binding proteins (IGFBPs) participate in the regulation of growth and differentiation of cells of the fibroblast lineage, possibly contributing to the fibrogenic process. The aim of this work was to study the expression and regulation of the IGF axis components in rat liver MF. Cultures of MF from passages 1 to 4 (P1-4) were studied. IGFBP secretion was analyzed by [(125)I]-IGF-I ligand and immunoblotting. IGF-I, IGF-IR, IGF-II/M6-PR, and IGFBP messenger RNA (mRNA) expression was assessed by Northern blot hybridization. DNA synthesis was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. MF from P1 to 4 constitutively expressed mRNA transcripts specific for IGF-I, IGF-IR, and IGF-II/M6-PR. In MF, biosynthesis of IGFBP-3 and -2 was observed that was stimulated by IGF-I, insulin, and transforming growth factor beta (TGF-beta), whereas platelet-derived growth factor (PDGF-BB) revealed inhibitory effects. IGF-I and to a lesser extent insulin increased DNA synthesis of MF. Simultaneous addition of recombinant human IGFBP-2 or -3 with IGF-I diminished the mitogenic effect of IGF-I on MF whereas preincubation of MF with IGFBP-2 or -3 further potentiated the IGF-I stimulated DNA synthesis. In conclusion, the present study demonstrates that the IGF axis may play a role in the regulation of MF proliferation in vitro which might be relevant in vivo for the process of fibrogenesis during acute and chronic liver injury.

Connective tissue growth factor induces c-fos gene activation and cell proliferation through p44/42 MAP kinase in primary rat hepatic stellate cells

Background/Aims Connective tissue growth factor (CCN2) is expressed during activation of hepatic stellate cells (HSC) and promotes HSC proliferation, adhesion, and collagen production. The aim of the study was to investigate CCN2 signaling pathways in HSC. Methods Primary HSC were obtained by enzymatic perfusion of rat liver. DNA synthesis was evaluated by [ 3H]thymidine incorporation. Phosphorylation of Elk-1, extracellular signal-regulated kinase (ERK1/2) and focal adhesion kinase (FAK) was evaluated by Western blot. Transcriptional factor binding activity was determined by gel mobility shift assay while c-fos promoter and CCN2 promoter activity was evaluated using luciferase reporters. c-fos mRNA expression was evaluated by Northern blot. Results CCN2 stimulated DNA synthesis and phosphorylation of FAK, Elk-1 and ERK1/2, the latter of which was blocked by heparin. The serum response element binding activity and luciferase reporter activity of the c-fos promoter, together with expression of c-fos, were enhanced by CCN2. CCN2-induced c-fos gene activation, expression and cell proliferation were blocked by inhibiting ERK1/2 with PD98059. CCN2 promoter activity was enhanced by TGF-β1 or PDGF via a Smad7-dependent pathway. Conclusions CCN2-stimulated HSC DNA synthesis is associated with transient induction of c-fos gene activation and expression as well as activation of the ERK1/2 signal pathway.

Angiotensin II stimulates DNA synthesis of rat pancreatic stellate cells by activating ERK through EGF receptor transactivation

Although angiotensin II (Ang II) is known to participate in pancreatic fibrosis, little is known as to the mechanism by which Ang II promotes pancreatic fibrosis. To elucidate the mechanism, we examined the action of Ang II on the proliferation of rat pancreatic stellate cells (PSCs) that play central roles in pancreatic fibrosis. Immunocytochemistry and Western blotting demonstrated that both Ang II type 1 and type 2 receptors were expressed in PSCs. [ 3H]Thymidine incorporation assay revealed that Ang II enhanced DNA synthesis in PSCs, which was blocked by Ang II type 1 receptor antagonist losartan. Western blotting using anti-phospho-epidermal growth factor (EGF) receptor and anti-phospho-extracellular signal regulated kinase (ERK) antibodies showed that Ang II-activated EGF receptor and ERK. Both EGF receptor kinase inhibitor AG1478 and MEK1 inhibitor PD98059 attenuated ERK activation and DNA synthesis enhanced by Ang II. These results indicate that Ang II stimulates PSC proliferation through EGF receptor transactivation—ERK activation pathway.

Comparative study of the short-term effects of a novel selective estrogen receptor modulator, ospemifene, and raloxifene and tamoxifen on rat uterus

To investigate the differential short-term effects of selective estrogen receptor (ER) modulators (SERMs) on uterus, we treated adult ovariectomized rats with a novel SERM, ospemifene (Osp), two previously established SERMs (tamoxifen and raloxifene (Ral)) and estradiol. The expression of two estrogen-regulated early response genes c-fos and vascular endothelial growth factor (VEGF), and DNA synthesis were analysed at 1–24 h after treatment of ovariectomized rats. Induction of c-fos mRNA by each of the SERMs showed a biphasic pattern with peaks at 3 and 20 h, respectively. The maximum level of VEGF mRNA was observed at 1 h after raloxifene and 6 h after tamoxifen or ospemifene treatment. Maximum levels of the c-fos and VEGF mRNA after raloxifene treatment were higher than those seen after treatments with E2 or a corresponding dose of tamoxifen or ospemifene. DNA synthesis was significantly increased by ospemifene, tamoxifen and raloxifene both in luminal and glandular epithelium. The stimulation was transient, peaking at 16 h. In comparison, the maximum level observed at 16 h after E2 treatment sustained at least until 24 h. DNA synthesis in stromal cells was increased by the SERMs but not by E2 at 24 h. When treated together with E2, the SERMs were able to antagonise E2-stimulated DNA synthesis at 16 h. Our results demonstrate that the initial response of uterus to ospemifene, raloxifene and tamoxifen includes activation of early response genes and even transient stimulation of DNA synthesis in spite of their different long-term effects. However, the early stimulatory events may be mediated by different mechanisms leading to diverging pathways in various tissue compartments and development of differential SERM-specific long-term responses of uterus.

Functional Analysis of Multiple Single-stranded DNA-binding Proteins from Methanosarcina acetivorans and Their Effects on DNA Synthesis by DNA Polymerase BI

Single-stranded DNA-binding proteins and their functional homologs, replication protein A, are essential components of cellular DNA replication, repair and recombination. We describe here the isolation and characterization of multiple replication protein A homologs, RPA1, RPA2, and RPA3, from the archaeon Methanosarcina acetivorans. RPA1 comprises four single-stranded DNA-binding domains, while RPA2 and RPA3 are each composed of two such domains and a zinc finger domain. Gel filtration analysis suggested that RPA1 exists as homotetramers and homodimers in solution, while RPA2 and RPA3 form only homodimers. Unlike the multiple RPA proteins found in other Archaea and eukaryotes, each of the M. acetivorans RPAs can act as a distinct single-stranded DNA-binding protein. Fluorescence resonance energy transfer and fluorescence polarization anisotropy studies revealed that the M. acetivorans RPAs bind to as few as 10 single-stranded DNA bases. However, more stable binding is achieved with single-stranded DNA of 18-23 bases, and for such substrates the estimated Kd was 3.82 +/- 0.28 nM, 173.6 +/- 105.17 nM, and 5.92 +/- 0.23 nM, for RPA1, RPA2, and RPA3, respectively. The architectures of the M. acetivorans RPAs are different from those of hitherto reported homologs. Thus, these proteins may represent novel forms of replication protein A. Most importantly, our results show that the three RPAs and their combinations highly stimulate the primer extension capacity of M. acetivorans DNA polymerase BI. Although bacterial SSB and eukaryotic RPA have been shown to stimulate DNA synthesis by their cognate DNA polymerases, our findings provide the first in vitro biochemical evidence for the conservation of this property in an archaeon.

Clinical and biologic relevance of hyaluronic acid and its fragments

I thank Dr. Slevin and coauthors for writing an excellent account of the clinical and biologic relevance of hyaluronic acid (HA) and its fragments. In 1985, the seminal work from Dr. Kumar's group offered a glimpse of the role of HA fragments in angiogenesis.1 In later years, the same group helped to elucidate some of the signal transduction mechanisms by which HA fragments stimulate bovine endothelial cell proliferation, migration and adhesion.2 Consistent with the findings of Dr. Kumar's group, we previously demonstrated that HA fragments of 10–15 disaccharide units induce proliferation and adhesion of human primary endothelial cells.3 Furthermore, primary cultures of human pulmonary artery (HPAEC), lung microvessel (HMVEC-L) and umbilical vein–derived (HUVEC) endothelial cells show differential responsiveness to angiogenic HA fragments. While angiogenic fragments stimulate DNA synthesis in HMVEC-L and HPAEC, HUVEC do not respond to angiogenic HA fragments.3 However, in contrast to the suggestion by Dr. Kumar's group that the biologic effects of angiogenic HA fragments and HA, observed in bovine endothelial cells, are mediated through cell surface CD44 receptors,4 we found that RHAMM is the functional cell surface HA receptor expressed in HPAEC and HMVEC-L. RHAMM–angiogenic HA fragment interaction induces tyrosine phosphorylation of p125FAK, paxillin and p42/44 ERK.3 It is likely that the angiogenic HA fragments stimulate proliferation of primary human endothelial cells through the MAP kinase pathway.3 Thus, it is possible that, in different cell types, angiogenic HA fragments mediate their biologic effects through different HA receptors. Regarding the findings of Dr. Kumar's group that, although HA levels are elevated in the sera of both Wilms' tumor patients and children with bone-metastasizing renal tumor of childhood (BMRTC), HA fragments are found only in the sera of BMRTC patients,5 our findings in cancers of the bladder, prostate and head/neck are in agreement.6, 7, 8 We found that HA levels are elevated in the urine of bladder cancer patients regardless of tumor grade [i.e., low (G1), intermediate (G2) and high (G3)]. However, angiogenic HA fragments are found only in the urine of patients with G2 or G3 bladder tumors.6 This finding could be explained by our observation that hyaluronidase levels are elevated only in G2 and G3 patients, which in turn could degrade tumor-associated HA into angiogenic fragments. We have also shown that in bladder tumor tissues HA is produced by both tumor stroma and tumor cells, whereas hyaluronidase (i.e., HYAL1 gene product) is produced only by tumor cells.9, 10 Also, we found that angiogenic HA fragments are present in high-grade prostate cancer tissues, which contain high levels of both HA and hyaluronidase.7 In our previous article, we demonstrated that both high and low m.w. HA (i.e., HA fragments) species are present in the saliva of high-stage head-and-neck squamous cell carcinoma patients but not in the saliva of normal individuals.8 Taken together, our findings and those of Dr. Kumar's group show that, in addition to the increased amounts of HA in cancer patients' body fluids (i.e., urine, serum and saliva) and tumor tissues, the size profile of HA species may be important in cancer diagnosis and for evaluating the biologic behavior of various tumors. Yours sincerely, Vinata Lokeshwar

Aging does not reduce the hepatocyte proliferative response of mice to the primary mitogen TCPOBOP.

It has been shown that the magnitude of DNA synthesis and the time at which maximal DNA synthesis occurs after two-thirds partial hepatectomy (PH) is greatly reduced in the liver of aged rodents compared to young animals. This reduction could represent an intrinsic defect in proliferation or a more specialized change in the response to PH. We therefore evaluated the proliferative capacity of hepatocytes in aged animals, following treatment with primary liver mitogens. We show that treatment of 12-month-old CD-1 mice with the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) caused an increase in hepatocyte proliferation similar to that seen in young (8-week-old) mice. The labeling index was 82% in the livers of aged mice versus 76% in young animals. Histological observation demonstrated that the number of hepatocytes entering mitoses was similar in both groups; the mitotic indices were 2.5 per thousand and 2.7 per thousand, respectively. Additional experiments showed that the timing of DNA synthesis and M phase were nearly identical in both aged and young mice. Stimulation of hepatocyte DNA synthesis was associated with increased expression of several cell cycle-associated proteins (cyclin D1, cyclin A, cyclin B1, E2F, pRb, and p107); all were comparable in aged mice and young mice. TCPOBOP treatment also increased expression of the Forkhead Box transcription factor m1b (Foxm1b) to a similar degree in both groups. In conclusion, hepatocytes retain their proliferative capacity in old age despite impaired liver regeneration. These findings suggest that therapeutic use of mitogens would alleviate the reduction in hepatocyte proliferation observed in the elderly.

Nutrient-dependent and Insulin-stimulated Phosphorylation of Insulin Receptor Substrate-1 on Serine 302 Correlates with Increased Insulin Signaling

Ser/Thr phosphorylation of insulin receptor substrate IRS-1 regulates insulin signaling, but the relevant phosphorylated residues and their potential functions during insulin-stimulated signal transduction are difficult to resolve. We used a sequence-specific polyclonal antibody directed against phosphorylated Ser(302) to study IRS-1-mediated signaling during insulin and insulin-like growth factor IGF-I stimulation. Insulin or IGF-I stimulated phosphorylation of Ser(302) in various cell backgrounds and in murine muscle. Wortmannin or rapamycin inhibited Ser(302) phosphorylation, and amino acids or glucose stimulated Ser(302) phosphorylation, suggesting a role for the mTOR cascade. The Ser(302) kinase associates with IRS-1 during immunoprecipitation, but its identity is unknown. The NH(2)-terminal c-Jun kinase did not phosphorylate Ser(302). Replacing Ser(302) with alanine significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and p85 binding and reduced insulin-stimulated phosphorylation of p70(S6K), ribosomal S6 protein, and 4E-BP1; however, this mutation had no effect on insulin-stimulated Akt or glycogen synthase kinase 3beta phosphorylation. Replacing Ser(302) with alanine reduced insulin/IGF-I-stimulated DNA synthesis. We conclude that Ser(302) phosphorylation integrates nutrient availability with insulin/IGF-I signaling to promote mitogenesis and cell growth.

Age-related changes in IGF-1 expression in submandibular glands of senescence-accelerated mice.

Saliva is known to play important roles in such functions as swallowing, mastication, speech, and taste. Furthermore, salivary glands synthesize and secrete a number of growth factors involved in cell/tissue homeostasis. It has been demonstrated that IGF-1, which is structurally analogous to insulin, has been shown to be expressed in mouse submandibular glands, and that IGF-1 stimulates DNA synthesis, amino acid uptake, protein synthesis, and glucose transport in various cells. Diminished function of the salivary glands is thought to lead to increased dental caries and periodontal diseases, which are commonly associated with aging. However, very little is known regarding the effects of age on IGF-1 expression in submandibular glands. The senescence-accelerated mouse (SAM), an experimental murine model of accelerated aging, has been extensively used to examine the mechanisms responsible for aging. In the present study, IGF-1 production and mRNA levels in the submandibular glands of SAM-P1 mice were examined. IGF-1 levels were determined by radioimmunoassay and IGF-1 mRNA levels by semi-quantitative RT-PCR. We found that IGF-1 protein levels in homogenates and IGF-1 mRNA levels decreased with age in SAMP1 mice. These findings suggest that IGF-1 synthesis in submandibular glands decreases with aging, and this may result in lower levels of cellular proliferation, regeneration and wound healing in aged oral tissues.

Serotonin and thromboxane A 2 stimulate platelet-derived microparticle-induced smooth muscle cell proliferation

Introduction At the sites of vascular injury, activated and aggregating platelets release small vesiculated structures called platelet microparticles (PMPs). Apart from PMPs they also release several vasoactive mediators including serotonin and thromboxane A 2 (TXA 2). PMPs, serotonin, and TXA 2 have been shown to stimulate vascular smooth muscle cell (VSMC) proliferation. Thus, this study is designed to examine the interaction between PMPs and serotonin or TXA 2 in inducing rabbit VSMC proliferation. Methods Growth-arrested rabbit SMCs were incubated in serum-free medium with different concentrations of PMPs with or without serotonin or TXA 2. VSMC proliferation was examined by increase in incorporation of [ 3H]thymidine into DNA and by increase in cell number. Results PMPs stimulated DNA synthesis in a dose-dependent manner; up to an added concentration of 30 μg/ml (1489±90%) they stimulated SMC proliferation in a logarithmic fashion. Serotonin at 50 μM (345±21%) and TXA 2 at 7.5 μM (900±36%) had their maximal effect. When added together, PMPs (10 μg/ml) and serotonin (5 μM), synergistically induced DNA synthesis (581±36% and 211±11% when added alone and 1201±95% when added together), whereas PMPs (10 μg/ml) and TXA 2 (5 μM) additively induced DNA synthesis (581±36% and 781±56% when added alone and 1262±115% when added together). These increases in DNA synthesis were paralleled by increase in cell number. Conclusion PMPs, serotonin, and TXA 2 are mitogenic to SMC, and function as amplification factors to each other, suggesting that inhibition of neointimal proliferation after vascular injury may require the combined use of multiple growth factor inhibitors to simultaneously block several critical cellular activation pathways.

High glucose blocks the effects of estradiol on human vascular cell growth: differential interaction with estradiol and raloxifene

Because diabetic women appear not to be protected by estrogen in terms of propensity to cardiovascular disease, we tested the possibility that chronic hyperglycemia modulates the effects of E 2 on vascular cell growth in vitro. Human endothelial cells (E304) and vascular smooth muscle cells (VSMC) were grown in normal glucose (5.5 mmol/l), high glucose (22 mmol/l) or high manitol (22 nmol/l; an osmotic control) for 7 days. In endothelial cells glucose per se stimulated DNA synthesis. However E 2- (but not RAL-) stimulated [ 3 H ] thymidine incorporation was attenuated in the presence of high glucose. In parallel, E 2-dependent MAP-kinase-kinase activity was blocked in the presence of high glucose. High glucose increased basal creatine kinase (CK) specific activity, but E 2-stimulated CK was not significantly impaired in the presence of high glucose. In VSMC, high glucose prevented the inhibitory effect of high E 2 (but not of high RAL) concentrations on DNA synthesis. High glucose also prevented E 2-induced MAP-kinase-kinase activity. In contrast, while high glucose augmented basal CK, the relative E 2-induced changes were roughly equal in normal and high high glucose media. Hence, high glucose blocks several effects of E 2 on vascular cell growth, which are mediated, in part, via the MAP-kinase system and are likely contributors to E 2’s anti-atherosclerotic properties. Since RAL’s estrogen-mimetic effects on human vascular cell growth were independent of MAP-kinase activation and were not affected by hyperglycemia, the potential use of RAL to circumvent the loss of estrogen function induced by hyperglycemia and diabetes in the human vasculature should be further explored.

Reduced Expression of P2Y1Receptors in Connexin43-Null Mice Alters Calcium Signaling and Migration of Neural Progenitor Cells

Glial calcium signals play important roles during CNS development. Calcium transients induced by ATP, acting on purinergic receptors, stimulate DNA synthesis, increase astrocytic and neural stem cell proliferation, and are prominent during the differentiation of radial glia. We have shown previously that expression of P2Y receptors in astrocytes is altered when connexin43 (Cx43) is downregulated. To evaluate the consequences of Cx43 deletion on calcium signaling during neural progenitor development, studies were performed on neurospheres derived from embryonic striatum. After adhesion, cells migrating from wild-type (WT) and Cx43-null neurospheres displayed spontaneous calcium oscillations. Such activity was blunted by apyrase, 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS-2179), and suramin, suggesting that ATP released by neural cells acts on purinergic receptors to induce calcium oscillations. The amplitudes of Ca2+ transients induced by P2Y but not P2X receptor agonists were larger in WT than in Cx43-null progenitors, suggesting that these two cell populations express different P2 receptors. Suramin, a nonselective P2 receptor antagonist, and MRS-2179, a P2Y1 receptor-selective antagonist, reduced the proliferation rate and the migration of WT progenitor cells to levels similar to those of Cx43-null cells. Conversely, exogenous expression of P2Y1 receptors in Cx43-null cells restored their migration pattern to levels seen in WT progenitors. However, treatment with P2 receptor antagonists did not alter the ratio of nestin to GFAP expression in WT neural progenitors. These data show that altered autocrine-paracrine communication attributable to reduced levels of P2Y1 receptors in neural progenitor cells lacking Cx43 affects proliferation and migration but not cell differentiation during early CNS development.

Ethylene stimulates endoreduplication but inhibits cytokinesis in cucumber hypocotyl epidermis.

The effects of ethylene on cell division are generally considered inhibitory. In this study, we demonstrate that transient ethylene exposure, while suppressing cytokinesis, stimulates DNA synthesis. We monitored DNA synthesis and cytokinesis in the epidermis of cucumber (Cucumis sativus) hypocotyls, an organ whose post-germination development involves strictly limited cell division. During exposure to ethylene, DNA synthesis, assessed by the incorporation of the thymidine homolog 5-bromo-2'-deoxyuridine, was detected in 20% of the epidermal cells, whereas DNA synthesis was nearly undetectable in normal air. Cytofluorometric analysis of nuclei in affected cells showed an up to 8-fold increase in DNA content. During this time, new cell plate formation was not detected. However, shortly after ethylene was removed, DNA content was rapidly restored to 2C (diploid) levels in all cells, and new cell plate formation dramatically increased. These results demonstrate that ethylene promotes DNA synthesis and its endoreduplication but inhibits cytokinesis, thereby maintaining some cells in G2 phase.

Hepatocyte growth factor in hepatic allograft biopsies: an immunohistochemical study

Hepatocyte growth factor (HGF) is a multipotent growth factor that is a powerful stimulator of DNA synthesis in a variety of cell types, especially hepatocytes. This factor is produced by nonparenchymal cells in the liver, presumably Kupffer cells, Ito cells, and sinusoidal endothelial cells. Research has shown that HGF is involved in tissue regeneration, wound healing, normal tissue growth, tumor progression, embryogenesis, and tumor invasion. Elevated serum levels of HGF have been documented in patients with acute renal failure and in renal transplant recipients; however, the importance of HGF in liver transplantation is unknown. The aim of this study was to determine whether induction of HGF expression is associated with acute rejection in liver transplants. We assessed the presence and density of HGF in hepatic allograft biopsy specimens compared with those from a control group of healthy patients.

Growth factors and nonparenchymal cell conditioned media induce mitogenic responses in stable long-term adult rat hepatocyte cultures

Most prior studies have characterized hepatocyte proliferative responses in culture systems that do not express a stable differentiated phenotype. We investigated the DNA synthetic response of long-term stable hepatocyte cultures to growth factor stimulation as well as conditioning with nonparenchymal cells (NPCs). Primary rat hepatocytes were cultured on a single layer of collagen (h/C) or Matrigel (h/M), or in a collagen sandwich (C/h/C) or collagen–Matrigel sandwich (M/h/C). Hepatocytes were cultured for 7 days to allow phenotypic stabilization before growth factor addition, except for h/C cultures, which are unstable, where growth factors were added 1 day after seeding. Culture medium was supplemented with a mixture of hepatocyte, epidermal, and vascular endothelial growth factors and interleukin-6, either directly or after conditioning with NPCs for 24 h. Growth factors alone induced hepatocyte DNA synthesis, as measured via [3H]thymidine uptake, in the h/C, C/h/C, and M/h/C configurations. h/M exhibited very low levels of DNA synthesis. In the C/h/C and M/h/C configurations, the greatest stimulation was obtained using NPC-conditioned growth factors. This response was sustained for several days and without decreasing albumin or urea synthesis. These results suggest that hepatocyte mitogens and NPC-derived factors can stimulate DNA synthesis in stable and differentiated hepatocyte cultures.

PDGF stimulates DNA synthesis in human vascular smooth muscle cells via a novel wortmannin-insensitive phosphatidylinositol 3-kinase

The class 1 A phosphatidylinositol 3-kinase enzymes consist of a number of heterodimeric complexes of regulatory and catalytic subunits and have been implicated in a number of cellular responses. While platelet-derived growth factor (PDGF)-induced chemotaxis of human vascular smooth muscle cells (HVSMC) is inhibited by both wortmannin and LY294002, DNA synthesis is only inhibited by LY294002. Serum-induced DNA synthesis however is inhibited by LY294002, wortmannin and rapamycin. Similarly PDGF-induced protein kinase B (PKB) activation is inhibited by LY294002 but not by wortmannin or rapamycin. In conclusion PDGF-induced DNA synthesis appears to occur through a phosphatidylinositol 3-kinase (PI3-K)-dependent, but wortmannin-insensitive, PKB/Akt pathway.

Sphingosylphosphorylcholine is a Melanogenic Stimulator for Human Melanocytes.

As lysosphingolipids have multiple bio-modulator functions in various types of cells, we measured the biological effects of sphingosylphosphorylcholine (SPC) on cultured human melanocytes to determine whether these lysosphingolipids have the potential to activate these cells. The addition of SPC to cultured human melanocytes significantly stimulated DNA synthesis assessed by [3H]thymidine and melanogenesis assessed by the release of [3H]H2O (tyrosinase activity), the incorporation of [14C]thiouracil (melanin synthesis) and dopa-oxidase activity. Reverse transcription-polymerase chain reaction of RNA isolated from human melanocytes exposed to SPC revealed an upregulation of mRNA transcripts for tyrosinase, microphthalmia-associated transcription factor-M, endothelin B receptor and the stem cell factor receptor, c-kit. An increase in expression of tyrosinase and c-kit proteins was also demonstrated by Western blot analysis. This stimulation of melanogenesis by SPC was associated with a marked increase in the phosphorylation of extracellular signal-regulated kinase 1/2. These results suggest that SPC may be a melanogenic stimulator of human melanocytes inducing the coordinated upregulated expression of various melanogenic molecules, including c-kit.

NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.

Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II-induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen alpha1(I) mRNA expression, and secretion of TGF-beta1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox-/- mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox-/- mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle alpha-actin and expression of TGF-beta1 were reduced in p47phox-/- mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis.

Regulation of Vascular Smooth Muscle Cell Responses to Insulin-like Growth Factor (IGF)-I by Local IGF-binding Proteins

Insulin-like growth factor (IGF)-I is a pleiotropic hormone that regulates vascular smooth muscle cell (VSMC) migration, proliferation, apoptosis, and differentiation. These actions are mediated by the IGF-I receptor. How activation of the same receptor by the same ligand leads to these diverse cellular responses is not well understood. Here we describe a novel mechanism specifying VSMC responses to IGF-I stimulation, distinctive for the pivotal roles of local IGF-binding proteins (IGFBPs). The role of local IGFBPs was indicated by comparing the activities of IGF-I and des-1-3-IGF-I, an IGF-I analog with reduced binding affinity to IGFBPs. Compared with IGF-I, des-1-3-IGF-I was more potent in stimulating DNA synthesis but much less potent in inducing directed migration of VSMCs. When the effects of individual IGFBPs were tested, IGFBP-2 and IGFBP-4 were found to inhibit IGF-I-stimulated DNA synthesis and migration. IGFBP-5 had an inhibitory effect on IGF-I-stimulated DNA synthesis, but it strongly potentiated IGF-I-induced VSMC migration. By using a non-IGF-binding IGFBP-5 mutant and an IGF-I-neutralizing antibody, it was demonstrated that IGFBP-5 also stimulates VSMC migration in an IGF-independent manner. This effect of IGFBP-5 was inhibited by soluble heparin and by treating cells with heparinase. Mutation of the heparin-binding motif of IGFBP-5 reduced its migration promoting activity. These findings suggest that local IGFBPs are important determinants of cellular responses to IGF-I stimulation, and a key player in this paradigm is IGFBP-5. IGFBP-5 not only modulates IGF-I actions, but it also stimulates cell migration by interacting with cell-surface heparan sulfate proteoglycans.

Involvement of mast cell chymase in bleomycin-induced pulmonary fibrosis in mice

The possible role of mast cell chymase in organ fibrosis was examined using a bleomycin-induced pulmonary fibrosis model in mice. Intratracheal injection of bleomycin to mice significantly increased not only hydroxyproline content but also chymase activity in the lung. Administration of a chymase inhibitor SUN C8077 (7-chloro-3-(3-amynophenyl) quinazoline-2, 4-dione methanesulfonate) dose-dependently reversed the bleomycin-induced increase in hydroxyproline content as well as chymase activity in the lung. Human chymase digested latent transforming growth factor-β1 (TGF-β1) to form mature TGF-β1 in vitro, which was inhibited by SUN C8077. Human chymase, on the other hand, failed to stimulate DNA synthesis of human lung fibroblasts CCD-8Lu and LL97A. Taken together, it is suggested that mast cell chymase might participate in the pathogenesis of pulmonary fibrosis, and that the chymase-induced fibrosis might be mediated at least in part by TGF-β1. Chymase inhibitor may be promising for treatment of pulmonary fibrosis in humans.