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Biomedical subjects

C Basilico

Publications and source records attributed to C Basilico.

At least 19 recordsLinked to original sources
PubMed2001

Evolution of coinfection with human immunodeficiency virus and hepatitis C virus in patients treated with highly active antiretroviral therapy.

A retrospective analysis of data from a cohort of patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) who were treated with highly active antiretroviral therapy (HAART) at 3 infectious diseases units in northern Italy was performed. While the patients were receiving HAART, CD4(+) cell counts significantly increased and HIV RNA serum levels decreased. However, no significant overall changes in alanine aminotransferase (ALT) levels and HCV RNA serum levels were observed. Fifteen (4.6%) of 323 patients died within 3 years of follow-up; death was related to cirrhosis in 5 patients (1.6%). No significant difference was observed between cirrhosis-related mortality and mortality related to other causes. Patients with ALT levels >4 times the normal values at initiation of HAART showed a significant decrease in ALT levels, whereas patients with normal ALT levels at initiation of HAART showed a significant increase over time, suggesting that HAART may have long-term beneficial or detrimental effects, depending on patient characteristics.

Alanine Transaminase↗
PubMed2001

Coevolution of HMG domains and homeodomains and the generation of transcriptional regulation by Sox/POU complexes.

The highly conserved homeodomains and HMG domains are components of a large number of proteins that play a role in the transcriptional regulation of gene expression during embryogenesis. Both the HMG domain and the homeodomain serve as interfaces for factor interactions with DNA, as well as with other proteins, and it is likely that the high degree of structural and sequence conservation within these domains reflects the conservation of basic aspects of these interactions. Classical HMG domain proteins have an ancient origin, being found in all eukaryotes, and are thought to have given rise to the metazoan-specific class of HMG domain proteins called the Sox proteins. Similarly, the metazoan-specific POU domain proteins are thought to have arisen from genes encoding ancestral homeodomain proteins. In this review, we summarize several examples of different HMG-homeodomain interactions that illustrate not only the ancient origin of each of these protein families, but also their relationship to each other, and discuss how coevolution of HMG and homeodomains may have lead to creation of the specialized Sox/POU protein complexes. Using the FGF-4 gene as an example, we also speculate on how coevolution of regulatory Sox/POU target DNA sequences may have occurred, and how the summation of these changes may have lead to the emergence of new developmental pathways.

Amino Acid Sequence↗
PubMed2001

Identification of receptor and heparin binding sites in fibroblast growth factor 4 by structure-based mutagenesis.

Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4. To begin to explore the structural determinants for this differential affinity, we determined the crystal structure of FGF4 at a 1.8-A resolution. FGF4 adopts a beta-trefoil fold similar to other FGFs. To identify potential receptor and heparin binding sites in FGF4, a ternary FGF4-FGFR1-heparin model was constructed by superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin structure. Mutation of several key residues in FGF4, observed to interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based on the modeling and mutational data, we propose that FGF4, like FGF2, but unlike FGF1, engages the betaC'-betaE loop in D3 and thus can differentiate between the IIIc and IIIb splice isoforms of FGFRs for binding. Moreover, we show that FGF4 needs to interact with both the 2-O- and 6-O-sulfates in heparin to exert its optimal biological activity.

3T3 Cells↗
PubMed2001

STAT1 mediates the increased apoptosis and reduced chondrocyte proliferation in mice overexpressing FGF2.

Unregulated FGF receptor signaling results in bone malformations that affect both endochondral and intramembranous ossification, and is the basis for several genetic forms of human dwarfism. FGF signaling inhibits chondrocyte proliferation and we have previously shown that the transcription factor STAT1 mediates the growth inhibitory effect of FGF in vitro. We provide genetic evidence that STAT1 is a modulator of the negative regulation of bone growth by FGF in vivo. We crossed Stat1(-/-) mice with a transgenic mouse line overexpressing human FGF2 (TgFGF). TgFGF mice exhibit phenotypes characterized by chondrodysplasia and macrocephaly, which affect endochondral and intramembranous ossification. We found that the chondrodysplasic phenotype of these mice results both from reduced proliferation and increased apoptosis of growth plate chondrocytes. Loss of STAT1 function in TgFGF mice led to a significant correction of the chondrodysplasic phenotype, but did not affect the skull malformations. The reduced proliferation of TgFGF growth plate chondrocytes, as well as their excessive apoptosis, were restored to near-normal levels in the absence of STAT1 function. Unregulated FGF signaling in TgFGF mice also induced apoptosis in calvarial osteoblasts that was not, however, corrected by the absence of STAT1. Detailed analysis of Stat1(-/-) growth plates uncovered a transient phenotype, characterized by an expansion of the proliferative zone and by acceleration of longitudinal bone growth, that attenuated as the animals grew older. These results document an essential role for STAT1 in FGF-mediated regulation of cell growth that is specific to the epiphyseal growth plate.

Animals↗
PubMed2000

Activation of fgf4 gene expression in the myotomes is regulated by myogenic bHLH factors and by sonic hedgehog.

The Fgf4 gene encodes an important signaling molecule which is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, we previously identified overlapping but distinct enhancer elements in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. Here we have investigated the hypothesis that Fgf4 is a target of myogenic bHLH factors. We show by mutational analysis that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. We show here that expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner.

3' Untranslated Regions↗
PubMed2000

Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer.

Fibroblast growth factor (FGF)-4 gene expression in the inner cell mass of the blastocyst and in EC cells requires the combined activity of two transcriptional regulators, Sox2 and Oct-3, which bind to adjacent sites on the FGF-4 enhancer DNA and synergistically activate transcription. Sox2 and Oct-3 bind cooperatively to the enhancer DNA through their DNA-binding, high mobility group and POU domains, respectively. These two domains, however, are not sufficient to activate transcription. We have analyzed a number of Sox2 and Oct-3 deletion mutants to identify the domains within each protein that contribute to the activity of the Sox2 x Oct-3 complex. Within Oct-3, we have identified two activation domains, the N-terminal AD1 and the C-terminal AD2, that play a role in the activity of the Sox2 x Oct-3 complex. AD1 also displays transcriptional activation functions in the absence of Sox2 while AD2 function was only detected within the Sox2 x Oct-3 complex. In Sox2, we have identified three activation domains within its C terminus: R1, R2, and R3. R1 and R2 can potentiate weak activation by Sox2 in the absence of Oct-3 but their deletion has no effect on the Sox2 x Oct-3 complex. In contrast, R3 function is only observed when Sox2 is complexed with Oct-3. In addition, analysis of Oct-1/Oct-3 chimeras indicates that the Oct-3 homeodomain also plays a critical role in the formation of a functional Sox2 x Oct-3 complex. Our results are consistent with a model in which the synergistic action of Sox2 and Oct-3 results from two major processes. Cooperative binding of the factors to the enhancer DNA, mediated by their binding domains, stably tethers each factor to DNA and increases the activity of intrinsic activation domains within each protein. Protein-protein and protein-DNA interactions then may lead to reciprocal conformational changes that expose latent activation domains within each protein. These findings define a mechanism that may also be utilized by other Sox x POU protein complexes in gene activation.

Base Sequence↗
PubMed2000

Signaling by fibroblast growth factors (FGF) and fibroblast growth factor receptor 2 (FGFR2)-activating mutations blocks mineralization and induces apoptosis in osteoblasts.

Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts.

Acrocephalosyndactylia↗
PubMed2000

Levels of the bcl-2 protein, fibronectin and alpha(5)beta(1) fibronectin receptor in HIV-1-infected patients with Kaposi's sarcoma.

Kaposi's sarcoma (KS) is an angioproliferative disease characterized by proliferation of neoplastic cells (spindle cells) mixed with endothelial and inflammatory cells. In this study we evaluated the role of the adhesive glycoprotein, fibronectin (FN) and its receptor alpha(5)beta(1) (FNR), and the proto-oncogene bcl-2, an anti-apoptotic protein. Significantly decreased serum levels of FN were noted in HIV-1-infected patients with KS, whereas serum levels of FNR were significantly increased in the same patients. Furthermore, increased FNR expression was observed on CD4 cells from KS patients. Serum levels of bcl-2 protein were significantly decreased in asymptomatic seropositive patients, whereas HIV-1-infected patients with KS showed increased serum levels of bcl-2. These results provide further information about interaction between integrins and the extracellular matrix and bcl-2 protein that can support cell survival either of neoplastic cells or endothelial and inflammatory cells.

AIDS-Related Opportunistic Infections↗
PubMed2000

Different point mutations in the met oncogene elicit distinct biological properties.

The MET proto-oncogene, encoding the tyrosine kinase receptor for HGF, controls genetic programs leading to cell growth, invasiveness, and protection from apoptosis. Recently, MET mutations have been identified in hereditary and sporadic forms of papillary renal carcinoma (PRC). Introduction of different naturally occurring mutations into the MET cDNA results in the acquisition of distinct biochemical and biological properties of transfected cells. Some mutations result in a high increase in tyrosine kinase activity and confer transforming ability in focus forming assays. These mutants hyperactivate the Ras signaling pathway. Other mutations are devoid of transforming potential but are effective in inducing protection from apoptosis and sustaining anchorage-independent growth. These Met(PRC) receptors interact more efficiently with the intracellular transducer Pi3Kinase. The reported results show that MET(PRC) mutations can be responsible for malignant transformation through different mechanisms, either by increasing the growth ability of cells or by protecting cells from apoptosis and allowing accumulation of other genetic lesions.-Giordano, S., Maffe, A., Williams, T. A., Artigiani, S., Gual, P., Bardelli, A., Basilico, C., Michieli, P., Comoglio, P. M. Different point mutations in the met oncogene elicit distinct biological properties.

Apoptosis↗
PubMed2000

Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice.

Fibroblast growth factor 1 (FGF1) and FGF2, the prototypic members of the FGF family of growth factors, have been implicated in a variety of physiological and pathological processes. Unlike most other FGFs, FGF1 and FGF2 are ubiquitously expressed and are not efficiently secreted. Gene knockouts in mice have previously demonstrated a role for FGF2 in brain development, blood pressure regulation, and wound healing. The relatively mild phenotypic defects associated with FGF2 deletion led to the hypothesis that the continued expression of other FGFs partially compensated for the absence of FGF2 in these mice. We now report our generation of mice lacking FGF1 and their use, in combination with our previously described FGF2 null mice, to produce mice lacking both FGF1 and FGF2. FGF1-FGF2 double-knockout mice are viable and fertile and do not display any gross phenotypic defects. In the double-knockout mice we observed defects that were similar in extent to those previously described for the FGF2 null mice. Differences in the organization of neurons of the frontal motor cortex and in the rates of wound healing were observed. We also observed in FGF2(-/-) mice and in FGF1-FGF2 double-knockout mice novel impairments in hematopoiesis that were similar in severity. Essentially no abnormalities were found in mice lacking only FGF1. Our results suggest that the relatively mild defects in FGF2 knockout animals are not a consequence of compensation by FGF1 and suggest highly restricted roles for both factors under normal developmental and physiological conditions.

Animals↗
PubMed1999

Mutant Met-mediated transformation is ligand-dependent and can be inhibited by HGF antagonists.

Mutations in the genes encoding for Met, Ret and Kit receptor tyrosine kinases invariably result in increased kinase activity and in the acquisition of transforming potential. However, the requirement of receptor ligands for the transformation process is still unclear. We have investigated the role of hepatocyte growth factor (HGF), the high-affinity ligand for Met, in mutant Met-mediated cell transformation. We provide evidence that the transforming potential displayed by mutant forms of Met found in human cancer is not only sensitive but entirely dependent on the presence of HGF, by showing that mutant Met transforms NIH3T3 fibroblasts, which produce endogenous HGF, but is not able to transform epithelial cells, unless exogenous HGF is supplied. Accordingly, mutant Met-induced transformation of NIH3T3 cells can be inhibited by HGF antagonists and increased by HGF stimulation. We also show that an engineered Met receptor which contains an oncogenic mutation but is impaired in its ability to bind HGF completely loses its transforming activity, which can be rescued by causing receptor dimerization using a monoclonal antibody. These results indicate that point mutations resulting in Met kinase activation are necessary but not sufficient to cause cell transformation, the latter being dependent on ligand-induced receptor dimerization. They also suggest that mutant Met-driven tumour growth depends on the availability and tissue distribution of active HGF, and provide proof-of-concept for the treatment of mutant-Met related pathologies by HGF-antagonizing drugs.

3T3 Cells↗
PubMed1999

FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway.

Several genetic forms of human dwarfism have been linked to activating mutations in FGF receptor 3, indicating that FGF signaling has a critical role in chondrocyte maturation and skeletal development. However, the mechanisms through which FGFs affect chondrocyte proliferation and differentiation remain poorly understood. We show here that activation of FGF signaling inhibits chondrocyte proliferation both in a rat chondrosarcoma (RCS) cell line and in primary murine chondrocytes. FGF treatment of RCS cells induces phosphorylation of STAT-1, its translocation to the nucleus, and an increase in the expression of the cell-cycle inhibitor p21WAF1/CIP1. We have used primary chondrocytes from STAT-1 knock-out mice to provide genetic evidence that STAT-1 function is required for the FGF mediated growth inhibition. Furthermore, FGF treatment of metatarsal rudiments from wild-type and STAT-1(-/-) murine embryos produces a drastic impairment of chondrocyte proliferation and bone development in wild-type, but not in STAT-1(-/-) rudiments. We propose that STAT-1 mediated down regulation of chondrocyte proliferation by FGF signaling is an homeostatic mechanism which ensures harmonious bone development and morphogenesis.

3T3 Cells↗
PubMed1999

Growth arrest-specific gene 6 (Gas6)/adhesion related kinase (Ark) signaling promotes gonadotropin-releasing hormone neuronal survival via extracellular signal-regulated kinase (ERK) and Akt.

We identified Ark, the mouse homolog of the receptor tyrosine kinase Axl (Ufo, Tyro7), in a screen for novel factors involved in GnRH neuronal migration by using differential-display PCR on cell lines derived at two windows during GnRH neuronal development. Ark is expressed in Gn10 GnRH cells, developed from a tumor in the olfactory area when GnRH neurons are migrating, but not in GT1-7 cells, derived from a tumor in the forebrain when GnRH neurons are postmigratory. Since Ark (Ax1) signaling protects from programmed cell death in fibroblasts, we hypothesized that it may play an antiapoptotic role in GnRH neurons. Gn10 (Ark positive) GnRH cells were more resistant to serum withdrawal-induced apoptosis than GT1-7 (Ark negative) cells, and this effect was augmented with the addition of Gas6, the Ark (Ax1) ligand. Gas6/Ark stimulated the extracellular signal-regulated kinase, ERK, and the serine-threonine kinase, Akt, a downstream component of the phosphoinositide 3-kinase (PI3-K) pathway. To determine whether ERK or Akt activation is required for the antiapoptotic effects of Gas6/Ark in GnRH neurons, cells were serum starved in the absence or presence of Gas6, with or without inhibitors of ERK and PI3-K signaling cascades. Gas6 rescued Gn10 cells from apoptosis, and this effect was blocked by coincubation of the cells with the mitogen-activated protein/ERK kinase (MEK) inhibitor, PD98059, or wortmannin (but not rapamycin). These data support an important role for Gas6/Ark signaling via the ERK and PI3-K (via Akt) pathways in the protection of GnRH neurons from programmed cell death across neuronal migration.

Androstadienes↗
PubMed1998

Distinct regulatory elements govern Fgf4 gene expression in the mouse blastocyst, myotomes, and developing limb.

Embryonic development requires a complex program of events which are directed by a number of signaling molecules whose expression must be rigorously regulated. We previously showed that expression of Fgf4, which plays an important role in postimplantation development and growth and patterning of the limb, is regulated in EC cells by the synergistic interaction of Sox2 and Oct-3 with the Fgf4 EC cell-specific enhancer. To verify whether this mechanism was also operating in vivo, and to identify new elements controlling Fgf4 gene expression in distinct developmental stages, we have analyzed the expression of LacZ reporter plasmids containing different fragments of the Fgf4 gene in transgenic mouse embryos. Utilizing these transgenic constructs we have been able to recapitulate, for the most part, Fgf4 gene expression during embryonic development. We show here that most of the cis-acting regulatory elements determining Fgf4 embryonic expression are located in conserved regions within the 3' UTR of the gene. The EC cell-specific enhancer is required to drive gene expression in the ICM of the blastocyst, and its activity requires the Sox and Oct-proteins binding sites. We were also able to identify specific and distinct enhancer elements that govern postimplantation expression in the somitic myotomes and the limb bud AER. The myotome-specific elements contain binding sites for bHLH myogenic regulatory factors, which appear to be essential for myotome expression. Finally, we present evidence that the very restricted pattern of expression of Fgf4 transcripts in the AER results from the combined action of positive and negative regulatory elements located 3' of the Fgf4 coding sequences. Thus the Fgf4 gene relies on multiple and distinct regulatory elements to achieve stage- and tissue-specific embryonic expression.

Animals↗
PubMed1998

Uncoupling signal transducers from oncogenic MET mutants abrogates cell transformation and inhibits invasive growth.

The assumption that genes encoding tyrosine kinase receptors could play a role in human cancers has been confirmed by the identification of oncogenic mutations in the kinase domain of RET and KIT. Recently, homologous residues were found mutated in MET, in papillary renal carcinomas (PRCs). The link coupling these genetic lesions to cellular transformation is still unclear. METPRC mutations result in increased kinase activity and-in some instances, i.e., M1250T substitution-in changes in substrate specificity. A direct correlation occurs between the transforming potential of METPRC mutants and their ability to constitutively associate with signal transducers through two phosphorylated tyrosines (Y1349VHVNATY1356VNV) located in the receptor tail. Substitution of these "docking tyrosines" with phenylalanines leaves unaffected the altered properties of the kinase but abrogates transformation and invasiveness in vitro. Uncoupling the receptor from signal transducers with a tyrosine-phosphorylated peptide derivative (YpVNV) inhibits invasive growth induced by METPRC mutants. These data indicate that constitutive receptor coupling to downstream signal transducers is a key mechanism in neoplastic transformation driven by mutated MET and suggest a therapeutic strategy to target neoplastic diseases associated with this oncogene.

Amino Acid Sequence↗
PubMed1998

Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2.

Basic fibroblast growth factor (FGF2) is a wide-spectrum mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels in many tissues and cell types and reaches high concentrations in brain and pituitary. FGF2 has been implicated in a multitude of physiological and pathological processes, including limb development, angiogenesis, wound healing, and tumor growth, but its physiological role is still unclear. To determine the function of FGF2 in vivo, we have generated FGF2 knockout mice, lacking all three FGF2 isoforms, by homologous recombination in embryonic stem cells. FGF2(-/-) mice are viable, fertile and phenotypically indistinguishable from FGF2(+/+) littermates by gross examination. However, abnormalities in the cytoarchitecture of the neocortex, most pronounced in the frontal motor-sensory area, can be detected by histological and immunohistochemical methods. A significant reduction in neuronal density is observed in most layers of the motor cortex in the FGF2(-/-) mice, with layer V being the most affected. Cell density is normal in other regions of the brain such as the striatum and the hippocampus. In addition, the healing of excisional skin wounds is delayed in mice lacking FGF2. These results indicate that FGF2, although not essential for embryonic development, plays a specific role in cortical neurogenesis and skin wound healing in mice, which, in spite of the apparent redundancy of FGF signaling, cannot be carried out by other FGF family members.

Animals↗