Mirella Gaboli

Immunocytochemical distribution of extracellular matrix receptors in human osteoclasts: A β3 integrin is colocalized with vinculin and talin in the podosomes of osteoclastoma giant cells

Human osteoclasts (OCLs) obtained from cell suspensions of surgically excised giant cell bone tumors (osteoclastomas) were attached to glass coverslips and analyzed by immunofluorescence with antibodies to integrins and cytoskeletal proteins. It was found that in OCLs (i) podosomes, identified by their F-actin core and by interference reflection microscopy, were predominantly found in a peripheral belt as described in avian OCLs; (ii) each F-actin core was surrounded by a ring of vinculin and talin; (iii) beta 1 integrin was diffuse in the ventral membrane; (iv) beta 3 integrin was distributed in intensely fluorescent rings surrounding F-actin cores; (v) beta 2 integrin was absent; (vi) beta 4 integrin was absent. The macrophages detected in the same coverslips displayed podosomes containing beta 2 but not beta 3, fibroblasts showed adhesion plaques positive for beta 1 and beta 3 but not for beta 2, and platelets were intensely positive for beta 3. These results indicate that OCLs produce an integrin complex that is absent in the monocyte-macrophage lineage.

Inhibition of cell proliferation by the interferon-inducible 204 gene, a member of the Ifi 200 cluster

The role of the IFN-inducible p204 as growth regulator was investigated by transfecting an expression vector constitutively expressing p204 into several cell lines. Like pRB and p107, p204 is a potent growth inhibitor in sensitive cells, as demonstrated by the cell focus assay. Since stable transfectants of sensitive lines constitutively overexpressing p204 could not be established in vitro, we inserted the 204 cDNA into a vector bearing an heavy-metal-inducible promoter. Here we show that proliferation of B6MEF fibroblasts lacking endogenous p204 is strongly inhibited by transient p204 expression in the nucleus. p204 delays G1 progression into the S-phase and cells accumulate with a DNA content equivalent to cells arrested in late G1. Moreover, the role of p204 in the control of cell growth in vivo was investigated by generating transgenic mice in which the Ifi 204 gene was constitutively expressed in all tissues. To this end, expression vectors bearing the 204 cDNA under the control of the SV40 viral promoter were constructed. The overexpression of the p204 transgene achieved by injecting fertilized mouse eggs with these vectors was compatible with embryo development up to the four-cell stage in an in vitro follow-up of 4.5 days. However, no viable animals with an intact copy of the transgene were obtained, suggesting that high and constitutive levels of p204 expression can impair normal embryo development. These findings indicate that p204 plays a negative role in growth regulation and provide new information about the molecular mechanisms exploited by IFNs to inhibit cell proliferation.

Beta3 Subunit of Vitronectin Receptor is Present in Osteoclast Adhesion Structures and Not in Other Monocyte-Macrophage Derived Cells

The distribution of extracellular matrix receptors in human osteoclasts has been studied; a beta 3 integrin is colocalized with vinculin and talin in the podosomes of osteoclastoma giant cells and not in macrophages from the same source.

Host genotype controls the ability of the ISGF3 complex to activate transcription of IFN‐inducible genes

C57BL/6 mice are unable to express the lfi 202 type genes upon injection in vivo of multiple dsRNA, poly rl:rC, or IFN‐treatment in vitro. For this purpose the 5′ terminal flanking region (called the b segment of 804 bp) was linked to a heterologous reporter gene chloramphenicol acetyl transferase (CAT) and transfected into NIH3T3 cells or BLK cells derived from the C57BL/6 strain. IFN‐α induced strong CAT activity in NIH3T3 but not in BLK cells. This lack of transcription activation was not due to a defect in STAT factor activity, since IFN‐α treatment in the presence of IFN‐γ priming induced translocation of the ISGF3 into the nucleus, and binding to the ISRE (IFN‐Stimulated Response Element) of the 202 gene even in C57BL/6 derived cells. Surprisingly when three tandem copies of the 202 ISRE (42 bp) were linked to a heterologous promoter (c‐fos promoter) driving the reporter CAT gene, activation was also observed in C57BL/6 cells upon IFN‐treatment. Finally, another IFN‐inducible gene, namely the Mx, was activated in C57BL/6 mice. Thus, the primary defect of the C57BL/6 strain leading to an impaired lfi 202 type gene response to IFN appears to be an inability of the ISGF3 complex to activate the endogenous promoter. Altogether these results suggest that unidentified nuclear factors related to the host genotype control the ability of the STAT factors to activate transcription upon IFN‐treatment.

Regulation of Gene Expression by Interferons

IFNs are a family of proteins produced by various cells following stimulation by biological or synthetic inducers. The interaction with the membrane receptor is followed by the activation of the expression of particular genes that are responsible for their antiviral, antiproliferative and immunomodulatory properties. Initiation of transcription is an early and critical event in the control of eukaryotic gene expression. In this review we will briefly discuss the mechanisms exploited by IFNs to control at transcriptional level the expression of inducible genes. In particular we will focus on some characteristics if cis-acting DNA elements that are located upstream from the initiation site for RNA transcription and of nuclear trans-acting factors that are required for modulation of gene expression by IFNs.

cAMP response element of murine cytomegalovirus immediate early gene enhancer is transactivated by ras oncogene products

Products of ras oncogenes strongly stimulate the activity of the reporter gene, chloramphenicol acetyltransferase (CAT), driven by a 1.2 kb fragment of the murine cytomegalovirus (MCMV) immediate early (IE) gene enhancer (pCMVCAT). To define the role of proteins binding to the unique cAMP response element (CRE) present in the IE enhancer, NIH 3T3 cells were cotransfected with prasZip6 plasmid, a mammalian expression vector containing a v-Ha-ras cDNA, together with p(delta)ACMVCAT (pCMVCAT without the CRE sequence). Lower stimulation of CAT activity was indeed observed upon deletion of the CRE sequence. Decreased levels of p(delta)ACMVCAT were also observed in cell lines carrying stably transfected ras oncogenes. Further support for the role of the CRE sequence in MCMV enhancer activation comes from the finding that v-Ha-ras expression increases the activity of a reporter gene, beta-galactosidase, driven by three tandem copies of CRE sequence about six-fold. Moreover, this transactivation was prevented by cotransfection of the dominant inhibitor mutant Ha-ras (Leu-61; Ser-186) and was not suppressed by cotransfection of Ha-ras (Asn-17), suggesting that the effect is due to activated ras protein, rather than normal p21ras. Finally the transactivation observed is accompanied by an increase in nuclear proteins binding to a labelled oligonucleotide homologous to the CRE sequence, as shown in a gel retardation assay. These results suggest that the CRE element contributes to the transactivation of the MCMV IE gene enhancer by ras oncogenes.