Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development, carcinogenesis, and lysosomal enzyme-related diseases.

Growth factor treatment enhances vestibular hair cell renewal and results in improved vestibular function

The vestibules of adult guinea pigs were lesioned with gentamicin and then treated with perilymphatic infusion of either of two growth factor mixtures (i.e., GF I or GF II). GF I contained transforming growth factor α (TGFα), insulin-like growth factor type one (IGF-1), and retinoic acid (RA), whereas GF II contained those three factors and brain-derived neurotrophic factor. Treatment with GF I significantly enhanced vestibular hair cell renewal in ototoxin-damaged utricles and the maturation of stereociliary bundle morphology. The addition of brain-derived neurotrophic factor to the GF II infusion mixture resulted in the return of type 1 vestibular hair cells in ototoxin-damaged cristae, and improved vestibular function. These results suggest that growth factor therapy may be an effective treatment for balance disorders that are the result of hair cell dysfunction and/or loss.

A kinase associated with chromatin that can be activated by ligand-p185c-Neu or epidermal growth factor-receptor interactions.

Some growth factors transduce positive growth signals, while others can act as growth inhibitors. Nuclear signaling events of previously quiescent cells stimulated with various growth factors have been studied by isolating the complexed chromatin-associated proteins and chromatin-associated proteins. Signals from the plasma membrane are integrated within the cells and quickly transduced to the nucleus. It is clear that several growth factors, such as epidermal growth factor, transforming growth factor alpha (but not transforming growth factor beta), and platelet-derived growth factor, utilize similar intracellular signaling biochemistries to modulate nucleosomal characteristics. The very rapid and consistent phosphorylation of nuclear p33, p54, and low molecular mass proteins in the range of 15-18 kDa after growth factor stimulation implies that there is a coordination and integration of the cellular signaling processes. Additionally, phosphorylation of p33 and some low molecular mass histones has been found to occur within 5 min of growth factor treatment and to reach a maximum by 30 min. In this study, we report that Neu receptor activating factor also utilizes the same signaling mechanism and causes p33 to become phosphorylated. In addition, both the tumor promoter okadaic acid (which inhibits protein phosphatases 1 and 2A) and phorbol ester (phorbol 12-tetradecanoate 13-acetate) stimulate phosphorylation of p33, p54, and low molecular mass histones. However, transforming growth factor beta, which is a growth inhibitor for fibroblasts, fails to increase p33 phosphorylation. In general, p33 phosphorylation patterns correspond to positive and negative mitogenic signal transduction. p33 isolated from the complexed chromatin-associated protein fraction appears to be a kinase, or tightly associated with a kinase, and shares antigenicity with the cell division cycle-dependent Cdk2 kinase as determined by antibody-dependent analysis. The rapid phosphorylation of nucleosomal proteins may influence sets of early genes needed for the induction and progression of the cell cycle.

Steatosis in chronic hepatitis C: Association with increased messenger RNA expression of collagen I, tumor necrosis factor-α and cytochrome P450 2E1

Background: Increased levels of tumor necrosis factor (TNF)-alpha and oxidative stress have been implicated as factors contributing to hepatic injury in fatty liver diseases. As steatosis is associated with an accelerated progression of fibrosis in chronic hepatitis C (HCV), we hypothesized that the messenger (m)RNA expression of genes involved with the production of reactive oxygen species, inflammation and cellular injury would be increased in liver tissue from subjects with steatosis and chronic HCV. Methods: Real-time polymerase chain reaction was performed to determine relative mRNA expression levels of collagen I, TNF-alpha, cytochrome P450 2E1 (CYP 2E1), transforming growth factor-beta1 and CD14 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation. Results: Multivariate analysis demonstrated that collagen I mRNA expression was increased by 199% in steatosis (P = 0.02), 85% in moderate to severe fibrosis (P = 0.02) and 157% in inflammation (P = 0.03). Livers of patients with steatosis also had an increase in TNF-alpha mRNA expression by 50% (P = 0.03) and CYP 2E1 expression by 37% (P = 0.04) compared with non-steatotic livers. Tumor necrosis factor-alpha protein was localized to Kupffer cells, bile ducts and portal inflammatory cells by immunohistochemistry. Conclusion: Increased expression of TNF-alpha may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV. (C) 2003 Blackwell Publishing Asia Pty Ltd.

Receptor regulation of phosphoinositide 3-hydroxykinase in the NG115-401L-C3 neuronal cell line: stimulation by insulin-like growth factor-I

The activation of phosphoinositide 3-hydroxykinase (P13K) is currently believed to represent the critical regulatory event which leads to the production of a novel intracellular signal. We have examined the control of this pathway by a number of cell-surface receptors in NG115-401L-C3 neuronal cells. Insulin-like growth factor-I stimulated the accumulation of 3-phosphorylated inositol lipids in intact cells and the appearance of P13K in antiphosphotyrosine-antibody-directed immunoprecipitates prepared from lysed cells, suggesting that P13K had been activated by a mechanism involving a protein tyrosine kinase. In contrast, P13K in these cells was not regulated by a variety of G-protein-coupled receptors, nerve growth factor acting via a low affinity receptor, or receptors for transforming growth factor-beta and interleukin-1. The receptor-specificity of P13K activation in these cells places significant constraints on the possible physiological function(s) of this pathway.

Epidermal Growth Factor Impairs Palatal Shelf Adhesion and Fusion in the T gf-β3 Null Mutant

The cleft palate presented by transforming growth factor-β3 (Tgf-β3 ) null mutant mice is caused by altered palatal shelf adhesion, cell proliferation, epithelial-to-mesenchymal transformation and cell death. The expression of epidermal growth factor (EGF), transforming growth factor-β1 ( Tgf-β1 ) and muscle segment homeobox-1 (Msx-1) is modified in the palates of these knockout mice, and the cell proliferation defect is caused by the change in EGF expression. In this study, we aimed to determine whether this change in EGF expression has any effect on the other mechanisms altered in Tgf-β 3 knockout mouse palates. We tested the effect of inhibiting EGF activity in vitro in the knockout palates via the addition of Tyrphostin AG 1478. We also investigated possible interactions between EGF, Tgf-β 1 and Msx-1 in Tgf-β 3 null mouse palate cultures. The results show that the inhibition of EGF activity in Tgf-β 3 null mouse palate cultures improves palatal shelf adhesion and fusion, with a particular effect on cell death, and restores the normal distribution pattern of Msx-1 in the palatal esenchyme. Inhibition of TGF-β 1 does not affect either EGF or Msx-1 expression.

Multiple expression control mechanisms of peroxisome proliferator-activated receptors and their target genes.

The peroxisome proliferator-activated receptors (PPAR) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. As ligand-activated receptors, they form a functional transcriptional unit upon heterodimerization with retinoid X receptors (RXRs). PPARs are activated by fatty acids and their derivatives, whereas RXR is activated by 9-cis retinoic acid. This heterodimer binds to peroxisome proliferator response elements (PPRE) residing in target genes and stimulates their expression. Recent reports now indicate that PPARs and RXRs can function independently, in the absence of a hetero-partner, to modulate gene expression. Of importance, these non-canonical mechanisms underscore the impact of both cofactors and DNA on gene expression. Furthermore, these different mechanisms reveal the increasing repertoire of PPAR 'target' genes that now encompasses non-PPREs containing genes. It is also becoming apparent that understanding the regulation of PPAR expression and activity, can itself have a significant influence on how the expression of subgroups of target genes is studied and integrated in current knowledge.

Prestress in the extracellular matrix sensitizes latent TGF-β1 for activation.

Integrin-mediated force application induces a conformational change in latent TGF-β1 that leads to the release of the active form of the growth factor from the extracellular matrix (ECM). Mechanical activation of TGF-β1 is currently understood as an acute process that depends on the contractile force of cells. However, we show that ECM remodeling, preceding the activation step, mechanically primes latent TGF-β1 akin to loading a mechanical spring. Cell-based assays and unique strain devices were used to produce a cell-derived ECM of controlled organization and prestrain. Mechanically conditioned ECM served as a substrate to measure the efficacy of TGF-β1 activation after cell contraction or direct force application using magnetic microbeads. The release of active TGF-β1 was always higher from prestrained ECM as compared with unorganized and/or relaxed ECM. The finding that ECM prestrain regulates the bioavailability of TGF-β1 is important to understand the context of diseases that involve excessive ECM remodeling, such as fibrosis or cancer.

Essential role of Smad3 in the inhibition of inflammation-induced PPARbeta/delta expression.

Wound healing proceeds by the concerted action of a variety of signals that have been well identified. However, the mechanisms integrating them and coordinating their effects are poorly known. Herein, we reveal how PPARbeta/delta (PPAR: peroxisome proliferator-activated receptor) follows a balanced pattern of expression controlled by a crosstalk between inflammatory cytokines and TGF-beta1. Whereas conditions that mimic the initial inflammatory events stimulate PPARbeta/delta expression, TGF-beta1/Smad3 suppresses this inflammation-induced PPARbeta/delta transcription, as seen in the late re-epithelialization/remodeling events. This TGF-beta1/Smad3 action involves an inhibitory effect on AP-1 activity and DNA binding that results in an inhibition of the AP-1-driven induction of the PPARbeta/delta promoter. As expected from these observations, wound biopsies from Smad3-null mice showed sustained PPARbeta expression as compared to those of their wild-type littermates. Together, these findings suggest a mechanism for setting the necessary balance between inflammatory signals, which trigger PPARbeta/delta expression, and TGF-beta1/Smad3 that governs the timely decrease of this expression as wound healing proceeds to completion.

Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias.

Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFβ-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFβ signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFβ signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes.

Amitriptyline aggravates the fibrosis process in a rat model of infravesical obstruction

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Combined effect of the finasteride and doxazosin on rat ventral prostate morphology and physiology

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cyclosporin but not tacrolimus significantly increases salivary cytokine contents in rats

Background: Cyclosporin (CsA) and tacrolimus (FK-506) are immunosuppressive drugs that specifically inhibit T-cell activation via calcineurin inhibition. Gingival overgrowth is a common side effect following the administration of CsA. The severity of gingival overgrowth seen in patients taking FK-506 is less than that observed with CsA. Little is known about the involvement of saliva in drug-induced gingival overgrowth. The purpose of this study was to investigate the salivary contents of tumor growth factor β1 (TGF-β1), epidermal growth factor (EGF), and interleukin-6 (IL-6) as well as the hystometry of gingival tissue obtained from rats treated with either FK-506 or CsA. Methods: For 30 or 60 days rats received daily subcutaneous injection doses of either CsA or FK-506 (10 mg/kg). The concentrations of TGF-β1, EGF, and IL-6 in saliva were determined by enzyme-linked immunosorbent assay, and after histological processing, the oral epithelium and connective tissue were assessed at the region of the lower first molars. Results: The levels of TGF-β1, EGF, and IL-6 in saliva were not significantly altered by any of the treatments after 30 days. After 60 days of treatment with CsA, gingival overgrowth and significant increase in salivary TGF-β1, EGF, and IL-6 concentrations were observed; no statistically significant changes were induced by FK-506. Conclusion: Within the limits of this experimental study, it can be concluded that CsA, but not FK-506, induced gingival overgrowth associated with an increase of the salivary levels of the cytokines TGF-β1, EGF, and IL-6.

Pro- and anti-inflammatory cytokines produced by human monocytes challenged in vitro with Paracoccidioides brasiliensis

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1β), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-β1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1β, IL-6, IL-10 and TNF-α. Pb18 induced higher levels of IL-1β, IL-6, and IL-10 than Pb265. IL-8 and TGF-β1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.

Pattern of macrophage activation in yersinia-resistant and yersinia-susceptible strains of mice

Th1 cells, in cooperation with activated macrophages, are required to overcome Yersinia enterocolitica infection in mice. The pathway macrophages utilize to metabolize arginine can alter the outcome of inflammation in different ways. The objective of this study was to verify the pattern of macrophages activation in Y. enterocolitica infection of BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice. Both strains of mice were infected with Y. enterocolitica O:8 WA 2707. Peritoneal macrophages and spleen cells were obtained on the 1st, 3rd and 5th day post-infection. The iNOS and the arginase activities were assayed in supernatants of macrophage cultures, by measuring their NO/citrulline and ornithine products, respectively. TGFβ-1 production was also assayed. The Th1 and Th2 responses were evaluated in supernatants of lymphocyte cultures, by IFN-γ and IL-4 production. Our results showed that in the early phase of Y. enterocolitica infection (1st and 3rd day), the macrophages from C57BL/6 mice produced higher levels of NO/citrulline and lower levels of ornithine than macrophages from BALB/c mice. The infection with Y. enterocolitica leads to an increase in the TGF-β1 and IL-4 production by BALB/c mice and to an increase in the IFN-γ levels produced by C57BL/6 mice. These results suggest that Y. enterocolitica infection leads to the modulation of M1 macrophages in C57Bl/6 mice, and M2 macrophages in BALB/c mice. The predominant macrophage population (M1 or M2) at the 1st and 3rd day of infection thus seems to be important in determining Y. enterocolitica susceptibility or resistance.