PED/PEA-15 interacts with the 67 kD laminin receptor and regulates cell adhesion, migration, proliferation and apoptosis.

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kD (PED/PEA-15) is an anti-apoptotic protein whose expression is increased in several human cancers. In addition to apoptosis, PED/PEA-15 is involved in the regulation of other major cellular functions, including cell adhesion, migration, proliferation and glucose metabolism. To further understand the functions of this protein, we performed a yeast two-hybrid screening using PED/PEA-15 as a bait and identified the 67 kD high-affinity laminin receptor (67LR) as an interacting partner. 67 kD laminin receptor is a non-integrin cell-surface receptor for the extracellular matrix (ECM), derived from the dimerization of a 37 kD cytosolic precursor (37LRP). The 67LR is highly expressed in human cancers and widely recognized as a molecular marker of metastatic aggressiveness. The molecular interaction of PED/PEA-15 with 67LR was confirmed by pull-down experiments with recombinant His-tagged 37LRP on lysates of PED/PEA-15 transfected HEK-293 cells. Further, overexpressed or endogenous PED/PEA-15 was co-immunoprecipitated with 67LR in PED/PEA-15-transfected HEK-293 cells and in U-373 glioblastoma cells, respectively. PED/PEA-15 overexpression significantly increased 67LR-mediated HEK-293 cell adhesion and migration to laminin that, in turn, determined PED/PEA-15 phosphorylation both in Ser-104 and Ser-116, thus enabling cell proliferation and resistance to apoptosis. PED/PEA-15 ability to induce cell responses to ECM-derived signals through interaction with 67LR may be of crucial importance for tumour cell survival in a poor microenvironment, thus favouring the metastatic spread and colonization.

High growth rate of benign thyroid nodules bearing RET/PTC rearrangements.

CONTEXT: Benign thyroid nodules display a broad range of behaviors from a stationary size to a progressive growth. The RET/PTC oncogene has been documented in a fraction of benign thyroid nodules, besides papillary thyroid carcinomas, and it might therefore influence their growth. OBJECTIVE: The aim of the present work was to evaluate whether RET/PTC in benign thyroid nodules associates with a different nodular growth rate. STUDY DESIGN: In this prospective multicentric study, 125 subjects with benign nodules were included. RET rearrangements were analyzed in cytology samples; clinical and ultrasonographic nodule characteristics were assessed at the start and at the end of the study. RESULTS: RET/PTC was present in 19 nodules. The difference between the mean baseline nodular volume of the RET/PTC- and RET/PTC+ nodules was not significant. After 36 months of follow-up, the RET/PTC+ group (n = 16) reached a volume higher than the RET/PTC- group (n = 90) (5.04 ± 2.67 vs. 3.04 ± 2.26 ml; P = 0.0028). We calculated the monthly change of nodule volumes as a percentage of baseline. After a mean follow-up of 36.6 months, the monthly volume increase of nodules bearing a RET rearrangement was 4.3-fold that of nodules with wild-type RET (1.83 ± 1.2 vs. 0.43 ± 1.0% of baseline volume; P < 0.0001). CONCLUSIONS: Benign thyroid nodules bearing RET rearrangements grow more rapidly than those with wild-type RET. Searching for RET rearrangements in benign thyroid nodules might be useful to the clinician in choosing the more appropriate and timely therapeutic option.

Sandblasted-acid-etched titanium surface influences in vitro the biological behavior of SaOS-2 human osteoblast-like cells.

Osseointegrated dental implants have been successfully used over the past several years, allowing functional replacement of missing teeth. Surface properties of titanium dental implants influence bone cell response. Implant topography appears to modulate cell growth and differentiation of osteoblasts thus affecting the bone healing process. Optimal roughness and superficial morphology are still controversial and need to be clearly defined. In the present study we evaluated in vitro the biological behavior of SaOS-2 cells, a human osteoblast-like cell line, cultured on two different titanium surfaces, smooth and sandblasted-acid-etched, by investigating cell morphology, adhesion, proliferation, expression of some bone differentiation markers and extracellular matrix components. Results showed that the surface topography may influence in vitro the phenotypical expression of human osteoblast-like cells. In particular the tested sandblasted-acid-etched titanium surface induced a significantly increased Co I deposition and α2-ß1 receptor expression as compared to the relatively smooth surface, promoting a probable tendency of SaOS-2 cells to shift toward a mature osteoblastic phenotype. It is therefore likely that specific surface properties of sandblasted-acid-etched titanium implants may modulate the biological behavior of osteoblasts during bone tissue healing.

The cross-talk between the urokinase receptor and fMLP receptors regulates the activity of the CXCR4 chemokine receptor.

The receptor (CXCR4) for the stromal-derived factor-1 (SDF1) and the urokinase-receptor (uPAR) are up-regulated in various tumors. We show that CXCR4-transfected cells migrate toward SDF1 on collagen (CG) and do not on vitronectin (VN). Co-expression of cell-surface uPAR, which is a VN receptor, impairs SDF1-induced migration on CG and allows migration on VN. Blocking fMLP receptors (fMLP-R), alpha-v integrins or the uPAR region capable to interact with fMLP-Rs, impairs migration of uPAR/CXCR4-transfected cells on VN and restores their migration on CG. uPAR co-expression also reduces the adherence of CXCR4-expressing cells to various components of the extracellular matrix (ECM) and influences the partitioning of beta1 and alpha-v integrins to membrane lipid-rafts, affecting ECM-dependent signaling. uPAR interference in CXCR4 activity has been confirmed in cells from prostate carcinoma. Our results demonstrate that uPAR expression regulates the adhesive and migratory ability of CXCR4-expressing cells through a mechanism involving fMLP receptors and alpha-v integrins.

Prevalence of RET/PTC rearrangement in benign and malignant thyroid nodules and its clinical application.

Fine-needle aspiration cytology (FNAC) is the primary means to distinguish benign thyroid nodules from malignant ones. About 20% of FNAC yields indeterminate results leading to unnecessary or delayed surgery. Many studies of tissue samples, the majority of which are retrospective advocate testing for RET rearrangements as a diagnostic adjunctive tool in thyroid nodules with indeterminate cytological findings. Because of the uncertain prevalence of RET rearrangements, its utility as a tumor marker is still controversial. The goal of this study was to establish the prevalence and the utility of testing for RET rearrangements in FNAC suspicious of cancer in a clinical setting. In this prospective study, we analysed a large series of thyroid aspirates by RT-PCR only and Southern blot on RT-PCR products for type 1 and 3 RET rearrangements. Results were compared with clinical findings, cytological diagnosis and final histopathology. By the higher sensitive Southern-blot on RT-PCR method, RET rearrangements were present in 36% of papillary thyroid carcinomas (RET/PTC-1, 12%; RET/PTC-3, 20%; both, 4%) and of 13.3% of benign nodules. By means of RT-PCR only, RET rearrangements were disclosed only in 14.3% of PTC and in 3.6% of benign nodules. No significant correlation was found between RET rearrangements and clinicopathological features of patients. These results indicate that molecular testing of thyroid nodules for RET/PTC must take into account of its high prevalence in benign nodules, inducing false positive diagnoses when the highly sensitive assay Southern-blot on RT-PCR is used. Its searching by means of RT-PCR only, has a specificity superior of conventional cytology and can be used to refine inconclusive FNAC.

Growing thyroid nodules with benign histology and RET rearrangement.

Some benign thyroid nodules are stationary in size over time while others grow progressively, indicating that there is a broad individual variability within benign nodules. To date, it is very difficult to predict if a benign thyroid nodule will grow in size and which will be its trend over time. While BRAF(V600E) is a highly specific marker of thyroid cancer, RET rearrangements have been disclosed also in non malignant thyroid lesions and their biological significance is debated. We compared the clinical history of three histologically benign thyroid nodules harboring RET rearrangements with that of 6 benign nodules bearing wild type RET. The nodules negative for RET rearrangements were followed for 10 years by ultrasonographic evaluation, showing a slow, constant enlargement. Three patients with benign nodules diagnosed at FNAC, were followed for 11, 9 and 7 years by annual ultrasonographic evaluation. After several years of latency, the nodules had an unexpected and gradual increase in their dimensions, reaching a large final size. A second FNAC confirmed the previous cytologic diagnosis of benign lesion. Because of the increasing size of the nodules, the patients were advised to surgery. Before undergoing thyroidectomy, we performed molecular diagnostic tests that revealed the absence of BRAF(V600E) and the presence of RET/PTC-1 in one nodule and RET/PTC-3 in the two others. Despite the presence of this oncogene, the samples were histologically classified as benign hyperplastic nodules. These findings lead us to speculate that histologically benign hyperplastic thyroid nodules containing RET rearrangements might represent a subgroup of nodules with a rapid size increase.

The Ca2+-calmodulin-dependent kinase II is activated in papillary thyroid carcinoma (PTC) and mediates cell proliferation stimulated by RET/PTC.

RET/papillary thyroid carcinoma (PTC), TRK-T, or activating mutations of Ras and BRaf are frequent genetic alterations in PTC, all leading to the activation of the extracellular-regulated kinase (Erk) cascade. The aim of this study was to investigate the role of calmodulin-dependent kinase II (CaMKII) in the signal transduction leading to Erk activation in PTC cells. In normal thyroid cells, CaMKII and Erk were in the inactive form in the absence of stimulation. In primary PTC cultures and in PTC cell lines harboring the oncogenes RET/PTC-1 or BRaf(V600E), CaMKII was active also in the absence of any stimulation. Inhibition of calmodulin or phospholipase C (PLC) attenuated the level of CaMKII activation. Expression of recombinant RET/PTC-3, BRaf(V600E), or Ras(V12) induced CaMKII activation. Inhibition of CaMKII attenuated Erk activation and DNA synthesis in thyroid papillary carcinoma (TPC-1), a cell line harboring RET/PTC-1, suggesting that CaMKII is a component of the Erk signal cascade in this cell line. In conclusion, PTCs contain an active PLC/Ca(2+)/calmodulin-dependent signal inducing constitutive activation of CaMKII. This kinase is activated by BRaf(V600E), oncogenic Ras, and by RET/PTC. CaMKII participates to the activation of the Erk pathway by oncogenic Ras and RET/PTC and contributes to their signal output, thus modulating tumor cell proliferation.

Insulin stimulates fibroblast proliferation through calcium-calmodulin-dependent kinase II.

Insulin effects are mediated by multiple integrated signals generated by the insulin receptor. Fibroblasts, as most of mammalian cells, are a target of insulin action and are important actors in the vascular pathogenesis of hyperinsulinemia. A role for calcium-calmodulin-dependent kinases (CaMK) in insulin signaling has been proposed but has been under investigated. We investigated the role of the CaMK isoform II in insulin signaling in human fibroblasts. A rapid and transient increase of intracellular calcium concentration was induced by insulin stimulation, followed by increase of CaMKII activity, via L type calcium channels. Concomitantly, insulin stimulation induced Raf-1 and ERK activation, followed by thymidine uptake. Inhibition of CaMKII abrogated the insulin-induced Raf-1 and ERK activation, resulting also in the inhibition of thymidine incorporation. These results demonstrate that in fibroblasts, insulin-activated CaMKII is necessary, together with Raf-1, for ERK activation and cell proliferation. This represents a novel mechanism in the control of insulin signals leading to fibroblast proliferation, as well as a putative site for pharmacological intervention.

Calcium-calmodulin-dependent kinase II (CaMKII) mediates insulin-stimulated proliferation and glucose uptake.

Cellular growth and glucose uptake are regulated by multiple signals generated by the insulin receptor. The mechanisms of individual modulation of these signals remain somewhat elusive. We investigated the role of CaMKII in insulin signalling in a rat skeletal muscle cell line, demonstrating that CaMKII modulates the insulin action on DNA synthesis and the negative feedback that down regulates glucose uptake. Insulin stimulation generated partly independent signals leading to the rapid activation of Akt, Erk-1/2 and CaMKII. Akt activation was followed by Glut-4 translocation to the plasma membrane and increase of glucose uptake. Then, IRS-1 was phosphorylated at S612, the IRS-1/p85PI3K complex was disrupted, Akt was no more phosphorylated and both Glut-4 translocation and glucose uptake were reduced. Inhibition of CaMKII abrogated the insulin-induced Erk-1/2 activation, DNA synthesis and phosphorylation of IRS-1 at S612. Inhibition of CaMKII also abrogated the down-regulation of insulin-stimulated Akt phosphorylation, Glut-4 membrane translocation and glucose uptake. These results demonstrate that: 1 - CaMKII modulates the insulin-induced Erk-1/2 activation and cell proliferation; 2 - after the initial stimulation of the IRS-1/Akt pathway, CaMKII mediates the down-regulation of stimulated glucose uptake. This represents a novel mechanism in the selective control of insulin signals, and a possible site for pharmacological intervention.

Calmodulin-dependent kinase IV links Toll-like receptor 4 signaling with survival pathway of activated dendritic cells.

Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin-dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4-/- DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.

Combined analysis of galectin-3 and BRAFV600E improves the accuracy of fine-needle aspiration biopsy with cytological findings suspicious for papillary thyroid carcinoma.

Ten to fifteen percent of fine-needle aspiration biopsy (FNAB) of thyroid nodules are indeterminate. Galectin-3 (Gal-3) and the oncogene BRAFV600E are markers of malignancy useful to improve FNAB accuracy. The objective of this study was to determine whether the combined analysis of Gal-3 and BRAFV600E expression in thyroid aspirates could improve the diagnosis in FNAB with suspicious cytological findings. Two hundred and sixty-one surgical thyroid tissues and one hundred and forty-four thyroid aspirates were analyzed for the presence of the two markers. In surgical specimens, Gal-3 expression was present in 27.4% benign nodules, 91.9% papillary (PTC) and 75% follicular (FTC) thyroid carcinomas. BRAFV600E was not detected in 127 benign nodules, as well as in 32 FTCs, while was found in 42.9% PTC. No correlation was found between BRAF mutation and Gal-3 expression. Forty-seven consecutive FNAB suspicious for PTC were analyzed for the presence of the two markers. Of these nodules, 23 were benign at histology, 6 were positive for Gal-3, none displayed BRAFV600E, and 17 were negative for both the markers. Twenty suspicious nodules were diagnosed as PTC and four FTCs at histology. Of these 24 carcinomas, 9 resulted positive for BRAFV600E, 17 for Gal-3, and 22 for one or both the markers. The sensitivity, specificity, and accuracy for the presence of Gal-3 and/or BRAFV600E were significantly higher than those obtained for the two markers alone. Notably, the negative predictive value increased from 70.8 to 89.5%. In conclusion, the combined detection of Gal-3 and BRAFV600E improves the diagnosis in FNAB with cytological findings suspicious for PTC and finds clinical application in selected cases.

Detection of RET/PTC, TRK and BRAF mutations in preoperative diagnosis of thyroid nodules with indeterminate cytological findings.

BACKGROUND: Fine-needle aspiration biopsy (FNAB) is the primary means to distinguish benign from malignant nodules and select patients for surgery. However, adjunctive diagnostic tests are needed because in 20-40% of cases the FNAB result is uncertain. OBJECTIVE: We investigated whether a search for the oncogenes RET/PTC, TRK and BRAF(V600E) in thyroid aspirates could refine an uncertain diagnosis. PATIENTS AND METHODS: A total of 132 thyroid aspirates, including colloid nodules, inadequate samplings, indeterminate and suspicious for malignancy were analysed by reverse transcription polymerase chain reaction (RT-PCR) and mutant allele-specific amplification techniques for the presence of oncogenes. RESULTS: No oncogenes were detected in 48 colloid nodules, 46 inadequate and 19 indeterminate FNABs, then confirmed to be benign at histology. No oncogenes were detected in one follicular thyroid cancer (FTC) with indeterminate cytology. Five out of six papillary thyroid cancers (83%) with FNAB suspicious for malignancy were correctly diagnosed by the presence of oncogenes. Among these, four (67%) contained the BRAF mutation and one (17%) contained RET/PTC-3. On final analysis, no false-positive results were reported in 131 samples and five out of seven carcinomas (71%) were correctly diagnosed. The finding of oncogenes in FNAB specimens suspicious for malignancy guided the extent of surgical resection, changing the surgery from diagnostic to therapeutic in five cases. CONCLUSIONS: Detection of RET/PTC, TRK and BRAF(V600E) in FNAB specimens is proposed as a diagnostic adjunctive tool in the evaluation of thyroid nodules with suspicious cytological findings.

In vivo activity of the cleaved form of soluble urokinase receptor: a new hematopoietic stem/progenitor cell mobilizer.

Cleaved forms of soluble urokinase receptor (c-suPAR) have been detected in body fluids from patients affected by various tumors. We recently reported increased c-suPAR levels in sera of healthy donors during granulocyte colony-stimulating factor (G-CSF)-induced mobilization of CD34(+) hematopoietic stem cells (HSC). In vitro, c-suPAR or its derived chemotactic peptide (uPAR(84-95)) stimulated migration of human CD34(+) HSCs and inactivated CXCR4, the chemokine receptor primarily responsible for HSC retention in bone marrow. These results suggested that c-suPAR could potentially contribute to regulate HSC trafficking from and to bone marrow. Therefore, we investigated uPAR(84-95) effects on mobilization of mouse CD34(+) hematopoietic stem/progenitor cells (HSC/HPC). We first showed that uPAR(84-95) stimulated in vitro dose-dependent migration of mouse CD34(+) M1 leukemia cells and inactivated murine CXCR4. uPAR(84-95) capability to induce mouse HSC/HPC release from bone marrow and migration into the circulation was then investigated in vivo. uPAR(84-95) i.p. administration induced rapid leukocytosis, which was associated with an increase in peripheral blood CD34(+) HSCs/HPCs. In vitro colony assays confirmed that uPAR(84-95) mobilized hematopoietic progenitors, showing an absolute increase in circulating colony-forming cells. uPAR(84-95) mobilizing activity was comparable to that of G-CSF; however, neither synergistic nor additive effect was observed in combining the two molecules. These findings show for the first time in vivo biological effects of c-suPAR. Its capability to mobilize HSCs suggests potential clinical applications in HSC transplantation.

The metastasis-associated 67-kDa laminin receptor is involved in G-CSF-induced hematopoietic stem cell mobilization.

The 67-kDa laminin receptor (67LR) is a nonintegrin cell-surface receptor with high affinity for laminin, which plays a key role in tumor invasion and metastasis. We investigated the role of 67LR in granulocyte colony-stimulating factor (G-CSF)-induced mobilization of CD34+ hematopoietic stem cells (HSCs) from 35 healthy donors. G-CSF-mobilized HSCs, including CD34+/CD38- cells, showed increased 67LR expression as compared with unstimulated marrow HSCs; noteworthy, also, is the fact that the level of 67LR expression in G-CSF-mobilized HSCs correlated significantly with mobilization efficiency. During G-CSF-induced HSC mobilization, the expression of laminin receptors switched from alpha6 integrins, which mediated laminin-dependent adhesion of steady-state human marrow HSCs, to 67LR, responsible for G-CSF-mobilized HSC adhesion and migration toward laminin. In vitro G-CSF treatment, alone or combined with exposure to marrow-derived endothelial cells, induced 67LR up-regulation in marrow HSCs; moreover, anti-67LR antibodies significantly inhibited transendothelial migration of G-CSF-stimulated marrow HSCs. Finally, G-CSF-induced mobilization in mice was associated with 67LR up-regulation both in circulating and marrow CD34+ cells, and anti-67LR antibodies significantly reduced HSC mobilization, providing the first in vivo evidence for 67LR involvement in stem-cell egress from bone marrow after G-CSF administration. In conclusion, 67LR up-regulation in G-CSF-mobilized HSCs correlates with their successful mobilization and reflects its increase in marrow HSCs, which contributes to the egress from bone marrow by mediating laminin-dependent cell adhesion and transendothelial migration.

Granulocyte Macrophage-Colony Stimulating Factor receptor expression on human cardiomyocytes from end-stage heart failure patients.

BACKGROUND: In remodelling ventricles, the progression of heart failure is associated with structural changes involving the extra-cellular matrix (ECM) and the cytoskeleton of cardiomyocytes, associated with fibrosis, cellular damage and death. The role of some cytokines and haematopoietic growth factors in the mechanism of both damage and regeneration of cardiac tissue during acute myocardial infarction has been demonstrated. Following heart damage, the development of scarred tissue was considered to be the only outcome, since myocytes were considered to be terminally differentiated cells. However, recent studies in animal models and adult human hearts have shown that myocytes can proliferate under the modulation of several factors. AIMS: To assess Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) receptor expression in healthy and diseased human hearts, and to evaluate the possible role of GM-CSF and its receptor in the regeneration of cardiac tissue in chronic cardiomyopathy. METHODS AND RESULTS: GM-CSFR expression in human cardiac tissue from explanted hearts of ten patients with end-stage heart failure and in cardiac biopsies from eight normal human hearts was studied by immunohistochemistry, and cellular and molecular biology assays. Our results demonstrated an increase in GM-CSFR in cardiomyocytes from end-stage heart failure tissues as compared to normal control tissues. CONCLUSIONS: We hypothesize that GM-CSF plays a role in apoptotic and/or ECM deposition processes as well as in cytoskeleton modification in the myocardium.

Detection of BRAF mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA).

OBJECTIVE: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAF(V600E)), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. DESIGN AND METHODS: We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. RESULTS: The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) follicular variants. No mutations of BRAF were detected in the normal thyroid tissues, nor in follicular adenomas or follicular carcinomas. No correlation was found between BRAF mutation and clinicopathologic features nor with recurrence during a postoperative follow-up period of 4-11 years. BRAF(V600E) significantly correlated with absence of node metastasis. CONCLUSIONS: BRAF(V600E) is present in PTC, both in the classic form and in follicular variant with similar prevalence. No correlation was found between BRAF mutation and aggressive clinical behavior. MASA-PCR proved to be a specific, sensitive and reliable method to detect BRAF T1799A in DNA extracted from different sources, including cytologic samples obtained either fresh or from archival glass slides. We propose this method as a useful tool to improve accuracy of preoperative diagnosis identifying PTC from biopsies with indeterminate cytologic findings.

Pendrin is a novel in vivo downstream target gene of the TTF-1/Nkx-2.1 homeodomain transcription factor in differentiated thyroid cells.

Thyroid transcription factor gene 1 (TTF-1) is a homeobox-containing gene involved in thyroid organogenesis. During early thyroid development, the homeobox gene Nkx-2.5 is expressed in thyroid precursor cells coincident with the appearance of TTF-1. The aim of this study was to investigate the molecular mechanisms underlying thyroid-specific gene expression. We show that the Nkx-2.5 C terminus interacts with the TTF-1 homeodomain and, moreover, that the expression of a dominant-negative Nkx-2.5 isoform (N188K) in thyroid cells reduces TTF-1-driven transcription by titrating TTF-1 away from its target DNA. This process reduced the expression of several thyroid-specific genes, including pendrin and thyroglobulin. Similarly, down-regulation of TTF-1 by RNA interference reduced the expression of both genes, whose promoters are sensitive to and directly associate with TTF-1 in the chromatin context. In conclusion, we demonstrate that pendrin and thyroglobulin are downstream targets in vivo of TTF-1, whose action is a prime factor in controlling thyroid differentiation in vivo.

Effect of granulocyte macrophage-colony stimulating factor on extracellular matrix deposition by dermal fibroblasts from patients with scleroderma.

OBJECTIVE: . To investigate the in vitro effect of granulocyte macrophage-colony stimulating factor (GM-CSF) on the deposition of extracellular matrix (ECM) in fibroblasts obtained from the skin of patients with systemic sclerosis (Ssc), compared to healthy controls. METHODS: Dermal fibroblasts obtained from 14 patients with SSc (7 with the diffuse form and 7 with CREST syndrome) and from 7 controls were studied. Both SSc and normal skin fibroblast cultures were stimulated for 4 and 8 days with 100 ng/ml GM-CFS. GM-CSF receptor (GM-CSFR) expression was determined by Western blot of cell lysates. Immunofluorescence was used to determine GM-CSFR expression and to investigate the deposition of ECM (type I collagen, fibronectin, and tenascin). Quantitative analysis of ECM was performed by ELISA. Expression of type I collagen and metalloproteinase 1 (MMP-1) mRNA was determined by real-time quantitative PCR. RESULTS: Deposition of ECM by normal fibroblasts appeared not to be influenced by stimulation with GM-CSF; in contrast, after stimulation with GM-CSF SSc fibroblasts showed increased deposition of fibronectin and tenascin, while type I collagen production was decreased; these results were found with both immunofluorescence and ELISA. Quantitative PCR revealed that GM-CSF inhibited the expression of mRNA type I collagen in SSc fibroblasts but not in normal fibroblasts, whereas levels of the main collagenolytic enzyme, MMP-1, were not affected. CONCLUSION: These results suggest that in SSc fibroblasts GM-CSF exerts a blocking effect on the deposition of type I collagen, through an inhibitory action on mRNA, while the production of other components of ECM such as fibronectin and tenascin is increased by stimulation with this cytokine.

Soluble and cleaved forms of the urokinase-receptor: degradation products or active molecules?

The urokinase-mediated plasminogen activation (PA) system is involved in many physiological and pathological events that include cell migration and tissue remodelling, such as embryogenesis, ovulation, inflammation, wound healing, angiogenesis, and tumor invasion and metastasis. The urokinase receptor (uPAR) is a key molecule of this system and can bind extracellular and cell membrane molecules such as urokinase (uPA), vitronectin (VN), integrins and chemotaxis receptors. These multiple interactions can be modulated by the shedding or the cleavage of the cell membrane receptor. Indeed, cleaved forms of uPAR, lacking the N-terminal D1 domain, have been detected on the surface of cells and in tissues, while soluble forms have been found in biological fluids. Cleaved and soluble forms could represent the intermediary products of the uPAR metabolism or active molecules with precise and distinct functional roles. Here, we review the data concerning the in vitro and in vivo identification of these uPAR forms, their origin and functions, and the role that uPAR shedding and cleavage could play in biological processes.