Pre-B-cell transcription factor 1 and steroidogenic factor 1 synergistically regulate adrenocortical growth and steroidogenesis

variety of transcription factors including Wilms tumor gene (Wt-1), steroidogenic factor 1 (Sf-1), dosage-sensitive sex reversal, adrenal hypoplasia congenita on the X-chromosome, Gene 1 (Dax-1), and pre-B-cell transcription factor 1 (Pbx1) have been defined as necessary for regular adrenocortical development. However, the role of Pbx1 for adrenal growth and function in the adult organism together with the molecular relationship between Pbx1 and these other transcription factors have not been characterized. We demonstrate that Pbx haploinsufficiency (Pbx1(+/-)) in mice is accompanied by a significant lower adrenal weight in adult animals compared with wild-type controls. Accordingly, baseline proliferating cell nuclear antigen levels are lower in Pbx1(+/-) mice, and unilateral adrenalectomy results in impaired contralateral compensatory adrenal growth, indicating a lower proliferative potential in the context of Pbx1 haploinsufficiency. In accordance with the key role of IGFs in adrenocortical proliferation and development, real-time RT-PCR demonstrates significant lower expression levels of the IGF-I receptor, and up-regulation of IGF binding protein-2. Functionally, Pbx1(+/-) mice display a blunted corticosterone response after ACTH stimulation coincident with lower adrenal expression of the ACTH receptor (melanocortin 2 receptor, Mc2-r). Mechanistically, in vitro studies reveal that Pbx1 and Sf-1 synergistically stimulates Mc2-r promoter activity. Moreover, Sf-1 directly activates the Pbx1 promoter activity in vitro and in vivo. Taken together, these studies provide evidence for a role of Pbx1 in the maintenance of a functional adrenal cortex mediated by synergistic actions of Pbx1 and Sf-1 in the transcriptional regulation of the critical effector of adrenocortical differentiation, the ACTH receptor.

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor

The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR(2)), which is required for post-endocytic sorting of PAR(2) to lysosomes, where degradation arrests signaling. The mechanisms of post-endocytic sorting of ubiquitinated receptors are incompletely understood. Here, we investigated the role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), in post-endocytic sorting and signaling of PAR(2). In HEK-PAR(2) cells, PAR(2) activating peptide (PAR(2)-AP) induced PAR(2) trafficking from the cell surface to early endosomes containing endogenous HRS, and then to lysosomes. HRS overexpression or knockdown with small interfering RNA caused formation of enlarged HRS-positive endosomes, where activated PAR(2) and c-Cbl accumulated, and PAR(2) failed to traffic to lysosomes. Overexpression of HRS prevented PAR(2)-AP-induced degradation of PAR(2), as determined by Western blotting. Overexpression of HRS mutant lacking an ubiquitin-binding motif similarly caused retention of PAR(2) in enlarged endosomes. Moreover, HRS overexpression or knockdown caused retention of ubiquitin-resistant PAR(2)Delta14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR(2)Delta14K/R. HRS overexpression or knockdown similarly prevented lysosomal trafficking and recycling of calcitonin receptor-like receptor, a non-ubiquitinated receptor that traffics to lysosomes after sustained activation and recycles after transient activation. Thus, HRS plays a critically important role in the post-endocytic sorting of single receptors, PAR(2) and CLR, to both degradative and recycling pathways. This sorting role for HRS is independent of its ubiquitin-interacting motif, and it can regulate trafficking of both ubiquitinated and non-ubiquitinated PAR(2) and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur downstream from HRS.

A functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production

Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. Here we report a microarray analysis showing that treatment of cultured bovine theca cells (TC) with BMP6 significantly (>2-fold; P<0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with CYP17A1 and other key transcripts involved in TC steroidogenesis including LHCGR, INHA, STAR, CYP11A1 and HSD3B1 also down-regulated. BMP6 also reduced expression of NR5A1 encoding steroidogenic factor-1 known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. RNAi-mediated knockdown of INSL3 reduced INSL3 mRNA and secreted protein level (75 and 94%, respectively) and elicited a 77% reduction in CYP17A1 mRNA level and 83% reduction in androstenedione secretion. Knockdown of RXFP2 also reduced CYP17A1 mRNA level (81%) and androstenedione secretion (88%). Conversely, treatment with exogenous (human) INSL3 increased androstenedione secretion ~2-fold. The CYP17 inhibitor abiraterone abolished androgen secretion and reduced expression of both INSL3 and RXFP2. Collectively, these findings indicate a positive autoregulatory role for INSL3 signaling in maintaining thecal androgen production, and visa versa. Moreover, BMP6-induced suppression of thecal androgen synthesis may be mediated, at least in part, by reduced INSL3-RXFP2 signaling.

Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets

Rationale: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor–bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown. Objective: We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets. Methods and Results: Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI–mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2–mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein–coupled receptors. In vivo, this selective (hem)immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2–induced G protein–coupled receptor signaling pathways. Conclusions: These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome.

Phenylephrine and endothelin-1 upregulate connective tissue growth factor in neonatal rat cardiac myocytes.

Cardiac hypertrophy is associated with hypertrophic growth of cardiac myocytes and increased fibrosis. Much is known of the stimuli which promote myocyte hypertrophy and the changes associated with the response, but the links between the two are largely unknown. Using subtractive hybridization, we identified three genes which are acutely (<1 h) upregulated in neonatal rat ventricular myocytes exposed to the alpha-adrenergic agonist, phenylephrine. One represented connective tissue growth factor (CTGF) which is implicated in fibrosis and promotes hypertrophy in other cells. We further examined the expression of CTGF mRNA and protein in cardiac myocytes using quantitative PCR and immunoblotting, confirming that phenylephrine increased CTGF mRNA (maximal within 1 h) and protein (increased over 4 - 24 h). Endothelin-1 promoted a greater, though transient, increase in CTGF mRNA, but the increase in CTGF protein was sustained over 8 h. Neither agonist increased CTGF mRNA in cardiac non-myocytes. By increasing the expression of CTGF in cardiac myocytes, hypertrophic agonists such as phenylephrine and endothelin-1 may promote fibrosis. CTGF may also propagate the hypertrophic response initiated by these agonists.

Evaluation of transformation growth factor beta(1), interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi

The aims of this study were to evaluate the immunomodulatory role of TGF-beta(1), 1L-10, and INF-gamma in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Aracatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n=15) and symptomatic (n=15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachex a, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-beta(1), IL-10, and INF-gamma. Naturally active in vitro produced TGF-beta(1) was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-beta(1) levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-gamma concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokire was lower in spleen extract. Although INF-gamma is being produced in canine infection, mean levels of TGF-beta(1) and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs A as predominantly type Th2. (c) 2006 Elsevier B.V. All rights reserved.

Fibroblast Growth Factor-2 Regulation of Sprouty and NR4A Genes in Bovine Ovarian Granulosa Cells

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

Toll-like receptor 2 knockdown modulates interleukin (IL)-6 and IL-8 but not stromal derived factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzymelinked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2. © 2013 Society for Reproduction and Fertility.

Prognostic value of vascular endothelial growth factor and hypoxia-inducible factor 1 alpha in canine malignant mammary tumors

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

Toll-like receptor 2 knockdown modulates Interleukin (IL)-6 and IL-8 but not Stromal Derived Factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

The relationship between lymphatic vascular density and vascular endothelial growth factor A (VEGF-A) expression with clinical-pathological features and survival in pancreatic adenocarcinomas

Abstract: Background Pancreatic cancer is a rare tumor with an extremely low survival rate. Its known risk factors include the chronic use of tobacco and excessive alcohol consumption and the presence of chronic inflammatory diseases, such as pancreatitis and type 2 diabetes. Angiogenesis and lymphangiogenesis, which have been the focus of recent research, are considered prognostic factors for cancer development. Knowing the angiogenic and lymphangiogenic profiles of a tumor may provide new insights for designing treatments according to the different properties of the tumor. The aim of this study was to evaluate the density of blood and lymphatic vessels, and the expression of VEGF-A, in pancreatic adenocarcinomas, as well as the relationship between blood and lymphatic vascular density and the prognostically important clinical-pathological features of pancreatic tumors. Methods Paraffin blocks containing tumor samples from 100 patients who were diagnosed with pancreatic cancer between 1990 and 2010 were used to construct a tissue microarray. VEGF expression was assessed in these samples by immunohistochemistry. To assess the lymphatic and vascular properties of the tumors, 63 cases that contained sufficient material were sectioned routinely. The sections were then stained with the D2-40 antibody to identify the lymphatic vessels and with a CD34 antibody to identify the blood vessels. The vessels were counted individually with the Leica Application Suite v4 program. All statistical analyses were performed using SPSS 18.0 (Chicago, IL, USA) software, and p values ≤ 0.05 were considered significant. Results In the Cox regression analysis, advanced age (p=0.03) and a history of type 2 diabetes (p=0.014) or chronic pancreatitis (p=0.02) were shown to be prognostic factors for pancreatic cancer. Blood vessel density (BVD) had no relationship with clinical-pathological features or death. Lymphatic vessel density (LVD) was inversely correlated with death (p=0.002), and by Kaplan-Meyer survival analysis, we found a significant association between low LVD (p=0.021), VEGF expression (p=0.023) and low patient survival. Conclusions Pancreatic carcinogenesis is related to a history of chronic inflammatory processes, such as type 2 diabetes and chronic pancreatitis. In pancreatic cancer development, lymphangiogenesis can be considered an early event that enables the dissemination of metastases. VEGF expression and low LVD can be considered as poor prognostic factors as tumors with this profile are fast growing and highly aggressive. Virtual slides. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5113892881028514

Biology of FGFRL1, the fifth fibroblast growth factor receptor

FGFRL1 (fibroblast growth factor receptor like 1) is the most recently discovered member of the FGFR family. It contains three extracellular Ig-like domains similar to the classical FGFRs, but it lacks the protein tyrosine kinase domain and instead contains a short intracellular tail with a peculiar histidine-rich motif. The gene for FGFRL1 is found in all metazoans from sea anemone to mammals. FGFRL1 binds to FGF ligands and heparin with high affinity. It exerts a negative effect on cell proliferation, but a positive effect on cell differentiation. Mice with a targeted deletion of the Fgfrl1 gene die perinatally due to alterations in their diaphragm. These mice also show bilateral kidney agenesis, suggesting an essential role for Fgfrl1 in kidney development. A human patient with a frameshift mutation exhibits craniosynostosis, arguing for an additional role of FGFRL1 during bone formation. FGFRL1 contributes to the complexity of the FGF signaling system.

Transforming growth factor-beta inhibits the expression of clock genes

Disturbances of sleep-wake rhythms are an important problem in Alzheimer's disease (AD). Circadian rhythms are regulated by clock genes. Transforming growth factor-beta (TGF-β) is overexpressed in neurons in AD and is the only cytokine that is increased in cerebrospinal fluid (CSF). Our data show that TGF-β2 inhibits the expression of the clock genes Period (Per)1, Per2, and Rev-erbα, and of the clock-controlled genes D-site albumin promoter binding protein (Dbp) and thyrotroph embryonic factor (Tef). However, our results showed that TGF-β2 did not alter the expression of brain and muscle Arnt-like protein-1 (Bmal1). The concentrations of TGF-β2 in the CSF of 2 of 16 AD patients and of 1 of 7 patients with mild cognitive impairment were in the dose range required to suppress the expression of clock genes. TGF-β2-induced dysregulation of clock genes may alter neuronal pathways, which may be causally related to abnormal sleep-wake rhythms in AD patients.