Mutant FGFR3 associated with SADDAN disease causes cytoskeleton disorganization through PLCγ1/Src-mediated paxillin hyperphosphorylation.

K650M/E substitutions in the Fibroblast growth factor receptor 3 (FGFR3) are associated with Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN) and Thanatophoric Dysplasia type II (TDII), respectively. Both SADDAN and TDII present with affected endochondral ossification marked by impaired chondrocyte functions and growth plate disorganization. In vitro, K650M/E substitutions confer FGFR3 constitutive kinase activity leading to impaired biosynthesis and accumulation of immature receptors in endoplasmic reticulum (ER)/Golgi. From those compartments, both SADDAN-FGFR3 and TDII-FGFR3 receptors engender uncontrolled signalling, activating PLCγ1, signal transducer and activator of transcription 1, 3 and 5 (STAT1/3/5) and ERK1/2 effectors. Here, we investigated the impact of SADDAN-FGFR3 and TDII-FGFR3 signalling on cytoskeletal organization. We report that SADDAN-FGFR3, but not TDII-FGFR3, affects F-actin organization by inducing tyrosine hyperphosphorylation of paxillin, a key regulator of focal adhesions and actin dynamics. Paxillin phosphorylation was upregulated at tyrosine 118, a functional target of Src and FAK kinases. By using Src-deficient cells and a Src kinase inhibitor, we established a role played by Src activation in paxillin hyperphosphorylation. Moreover, we found that SADDAN-FGFR3 induced FAK phosphorylation at tyrosines 576/577, suggesting its involvement as a Src co-activator in paxillin phosphorylation. Interestingly, paxillin hyperphosphorylation by SADDAN-FGFR3 caused paxillin mislocalization and partial co-localization with the mutant receptor. Finally, the SADDAN-FGFR3 double mutant unable to bind PLCγ1 failed to promote paxillin hyperphosphorylation, pointing to PLCγ1 as an early player in mediating paxillin alterations. Overall, our findings contribute to elucidate the molecular mechanism leading to cell dysfunctions caused by SADDAN-FGFR3 signalling.

Upon dendritic cell (DC) activation chemokines and chemokine receptor expression are rapidly regulated for recruitment and maintenance of DC at the inflammatory site.

Dendritic cells (DC) are highly motile antigen-presenting cells that are recruited to sites of infection and inflammation to antigen uptake and processing. Then, to initiate T cell-dependent immune responses, they migrate from non-lymphoid organs to lymph nodes and the spleen. Since chemokines have been involved in human DC recruitment, we investigated the role of chemokines on mouse DC migration using the mouse growth factor-dependent immature DC line (D1). In this study, we characterized receptor expression, responsiveness to chemoattractants and chemokine expression of D1 cells during the maturation process induced by lipopolysaccharide (LPS). MIP-1alpha and MIP-5 were found to be the most effective chemoattractants, CCR1 was the main receptor expressed and modulated during LPS treatment, and MIP-2, RANTES, IP-10 and MCP-1 were the chemokines modulated during DC maturation. Thus, murine DC respond to a unique set of CC and CXC chemokines, and the maturational stage determines the program of chemokine receptors and chemokines that are expressed. Since CCR1 is modulated during the early phases of DC maturation, our results indicate that the CCR1 receptor may participate in the recruitment and maintenance of DC at the inflammatory site.

Positive selection of apoptosis-resistant cells correlates with activation of dominant-negative STAT5.

The STAT5 activation has important roles in cell differentiation, cell cycle control, and development. However, the potential implications of STAT5 in the control of apoptosis remain unexplored. To evaluate any possible link between the erythropoietin receptor (EpoR) JAK2/STAT5 transduction pathway and apoptosis, we have investigated apoptosis-resistant cells (ApoR) that arose from positive selection of the erythroid-committed Ba/F3EpoR cells triggered to apoptosis by ectopic expression of the HOX-B8 homeotic gene. We show that JAK2 is normally activated by Epo in both Ba/F3EpoR and ApoR cells. In contrast, both STAT5a and STAT5b isoforms are uniquely activated in a C-truncated form (86 kDa) only in ApoR cells. Analysis of ApoR and Ba/F3EpoR subclones confirmed that the switch to the truncated STAT5 isoform coincides with apoptosis survival and that ApoR do not derive from preexisting cells with a shortened STAT5. In addition, ApoR cells die in the absence of Epo. This indicates that resistance to apoptosis is not because of a general defect in the apoptotic pathway of ApoR cells. Furthermore, we show that the 86-kDa STAT5 protein presents a dominant-negative (DN) character. We hypothesize that the switch to a DN STAT5 may be part of a mechanism that allows ApoR cells to be selectively advantaged during apoptosis. In conclusion, we provide evidence for a functional correlation between a naturally occurring DN STAT5 and a biological response.

Activation of the JAK/STAT pathway leads to proliferation of ST14A central nervous system progenitor cells.

We were interested in whether central nervous system progenitor cells possess the signal transduction machinery necessary to mediate cytokine functions and whether this machinery can become activated upon stable expression of a particular cytokine receptor. For this purpose we utilized a previously obtained conditionally immortalized striatum-derived nestin-positive cell line (ST14A). We found that ST14A cells express Jak2, but not Jak1 or Tyk2. An identical pattern of expression was found in embryonic striatal tissue. To evaluate the susceptibility of these cytokine specific cytoplasmic transducers to activation, ST14A cells were stably transfected with the alpha and beta (AIC2A) chains of the murine interleukin-3 receptor. Four independent lines expressing both the alpha and beta receptor subunits were obtained. We found that cells from each of these lines were induced to proliferate upon exposure to interleukin-3. Dose response curve, antibody blocking experiments and binding studies revealed that the response was mediated by the reconstituted high affinity interleukin-3 receptor. Immunoprecipitation studies on these cells showed that Jak2 and Stat5 were being phosphorylated after stimulation of the reconstituted receptor. These results indicate that members of the JAK/STAT family of proteins are expressed in central nervous system progenitor cells and are susceptible to activation through stimulation of an exogenously expressed cytokine receptor, ultimately leading to cell proliferation.

Erythropoietin receptor signals both proliferation and erythroid-specific differentiation.

Ectopic expression of the erythropoietin receptor (EPO-R) in Ba/F3, an interleukin 3-dependent progenitor cell line, confers EPO-dependent cell growth. To examine whether the introduced EPO-R could affect differentiation, we isolated Ba/F3-EPO-R subclones in interleukin 3 and assayed for the induction of beta-globin mRNA synthesis after exposure to EPO. Detection of beta-globin mRNA was observed within 3 days of EPO treatment, with peak levels accumulating after 10 days. When EPO was withdrawn, expression of beta-globin mRNA persisted in most clones, suggesting that commitment to erythroid differentiation had occurred. Although EPO-R expression also supports EPO-dependent proliferation of CTLL-2, a mature T-cell line, those cells did not produce globin transcripts, presumably because they lack requisite cellular factors involved in erythrocyte differentiation. We conclude that the EPO-R transmits signals important for both proliferation and differentiation along the erythroid lineage.

c-fos proto-oncogene transient transcription is negatively affected in the ELa4-2 transformed rat cell line.

To study the effects of the regulatory phosphoprotein ICP4 of the Herpes simplex virus, (HSV), a DNA tumor virus, on the induction of gene expression by the epidermal growth factor (EGF), we have constructed a cell line, ELa4-2, which constitutively expresses the a-4 gene product. The ELa4-2 cells are derived from the rat fibroblast EL2, in which EGF induces a marked c-fos and c-myc proto-oncogene transcription. Here we report that in ELa4-2 cells, the gene expression induced by EGF was negatively affected in respect to that obtained stimulating the parental EL2 cells. In particular, we studied the c-fos and c-myc proto-oncogene transcription induced by EGF. We found that in ELa4-2 cells the c-fos induction was dramatically reduced in comparison with the c-fos induction obtained in the parental EL2 cells. On the contrary, the c-myc induction by EGF was not affected by the presence of ICP4. Finally, we compared the HSV infectivity in ELa4-2 versus the EL2 cells. We showed that the virus growth capability was reduced, in the cells expressing ICP4.

Enhanced expression of interleukin-3 and granulocyte-macrophage colony-stimulating factor receptor subunits in murine hematopoietic cells stimulated with hematopoietic growth factors.

AIC2A and AIC2B are closely related genes encoding components of the receptors for murine interleukin-3 (IL-3) (AIC2A) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5 (AIC2B). We have studied the parallel regulation of expression of these genes in erythroid and myeloid progenitor cell lines. AIC2A and AIC2B transcription was transiently induced in these cells in response to a variety of hematopoietic growth factors, including erythropoietin (EPO), monocyte-CSF, IL-3, GM-CSF, and stem cell factor (SCF or kit ligand). Run-on assays established that the increase occurred mainly at the transcriptional level. Immunoprecipitation experiments confirmed that the increase in messenger RNA expression resulted in augmented synthesis of both AIC2A and AIC2B proteins, and binding studies further showed these proteins to be functional. We observed a fourfold increase in low-affinity IL-3 sites in an erythroid precursor cell line stimulated with EPO, and a threefold increase in GM-CSF high-affinity sites in a myeloid cell line stimulated with IL-3. In addition, we showed that the increase in the IL-3 receptor chain AIC2A in the erythroid precursor cell line correlated with the ability of IL-3 to exert a cooperative effect with EPO in the induction of beta-globin in these cells.

Epidermal growth factor induces, in the EL alpha 4-2 cell line, herpes simplex virus-1 alpha 4 gene transcription in the absence of the viral trans-activator VP16.

We have constructed a fibroblast cell line (EL alpha 4-2) which constitutively expresses the alpha 4 gene of herpes simplex virus type 1. We studied the induction of the alpha 4 gene, in the absence of the viral activator VP16, by stimulating EL alpha 4-2 cells with different growth factors. Here we report that a rapid, transient induction of the alpha 4 gene occurs only when EL alpha 4-2 fibroblasts are stimulated with purified epidermal growth factor (EGF). Such an induction does not require de novo protein synthesis. The role of cellular factors on the EGF-mediated induction of the alpha 4 gene has been analyzed by gel mobility shift assays using nuclear extracts from EL alpha 4-2 cells stimulated or not with EGF. The results obtained show that two factors bind to TAATGARAT (R = purine) regardless of EGF-stimulation. We conclude that a mechanism, different from the one involving VP16, is responsible for alpha 4 gene activation in EL alpha 4-2 cells and that the DNA-protein architecture is maintained at the TAATGARAT regulatory site regardless of changes in the transcriptional state induced by EGF.

Altered growth factor sensitivity in EL2 rat fibroblasts: influence of this biological characteristic on cell growth.

Extensive evidence indicate that platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) play a key role in the stimulation of the 3T3 fibroblast replication: in this connection, PDGF and EGF act as a competence and a progression factor, respectively. We have previously demonstrated that EGF alone leads density-arrested EL2 rat fibroblasts to synthesize DNA and proliferate in serum-free cultures. Here, we have analyzed the role of EGF in the control of EL2 cell proliferation. Our data show a dose-related effect of EGF on DNA synthesis and cell growth, with maximal stimulation for both parameters at 20 ng/ml. On the other hand, autocrine production of PDGF or PDGF-like substances by EL2 cells is seemingly excluded by experiments with anti-PDGF serum or medium conditioned by EL2 fibroblasts. EGF binding studies show that EL2 cells possess high affinity EGF receptors, at a density level 3 to 4-fold higher than other fibroblastic lines. In addition, EL2 cells show a normal down-regulation of EGF receptors, following exposure to EGF, but PDGF, fibroblast growth factor (FGF), transforming growth factor beta (TGF beta) and bombesin have not decreased the affinity of EGF receptor for its ligand. Moreover, in EL2 cells, the EGF is able to induce the synthesis of putative intracellular regulatory proteins that govern the PDGF-induced competence in 3T3 cells. Our data indicate that EGF in EL2 cells may act as both a competence and a progression factor, via induction of the mechanisms, regulated in other cell lines by cooperation between different growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Secreted proteins induced by epidermal growth factor and transforming growth factor beta in EL2 rat fibroblasts. Role in the mitogenic response.

Most growth active hormones and peptides are mitogenic only in the presence of other growth factors [e.g., Platelet Derived Growth Factor (PDGF) and Epidermal Growth Factor (EGF) in "competence-progression" fibroblast model]. We have previously described that EGF alone is able to induce the signals which appear necessary for the mitogenic stimulation of EL2 rat embryo fibroblast line. Recently, we have demonstrated that Transforming Growth Factor beta (TGF beta) slightly stimulates the mitogenic response in EL2 cells. Here, we show that in EGF-treated EL2 cells the induction of at least four inducible-secreted proteins (ISPs, range from 29,000 to 68,000 Mr) is accompanied by a marked increase in DNA synthesis. In contrast, TGF beta or different concentrations of EGF induce a slow increase of the ISPs proportional to slow induction in DNA synthesis. Our results suggest that the mitogenic response in EL2 cell line may be connected with the qualitative and quantitative induction of these secreted proteins.

TGF beta induces a sustained c-fos expression associated with stimulation or inhibition of cell growth in EL2 or NIH 3T3 fibroblasts.

We have previously indicated that epidermal growth factor (EGF) plays a fundamental role in the proliferation control of EL2 rat fibroblast line. It is shown here that transforming growth factor beta (TGF beta) stimulates both DNA synthesis and proliferation of EL2 cells, while exerting an inhibitory effect on the growth of murine NIH-3T3 fibroblasts. We also report the effect of TGF beta and EGF on c-fos expression in EL2 cells, as compared to that of TGF beta in NIH-3T3 fibroblasts. In EL2 cells EGF induces a transient c-fos expression at both mRNA and protein level, as previously observed in NIH-3T3 fibroblasts treated with platelet-derived or fibroblast growth factor (PDGF, FGF). Conversely, TGF beta induces in EL2 cells a sustained expression of fos mRNA and protein, which are still detectable at least 24 and 7 hr after treatment respectively. In NIH-3T3 fibroblasts TGF beta causes a sustained fos RNA expression, which is not associated, however, with detectable fos protein. We conclude that in fibroblasts stimulated by mitogens c-fos expression may be differentially modulated, depending of the growth factor and the cell line. This is seemingly due to differential regulation of fos gene expression, not only at the transcriptional and/or post-transcriptional level (transient or sustained fos RNA induction by EGF or TGF beta in EL2 cells), but also at the translational level (fos protein(s) induction by TGF beta in EL2 but not NIH-3T3 fibroblasts, possibly related to the stimulatory vs inhibitory effect of this factor on the growth of the former vs the latter line).

Role of the c-fos gene expression on the mitogenic response in EL2 rat fibroblasts.

Stimulation of the growth of quiescent fibroblasts by polypeptide growth factors is accompanied by the rapid induction of the c-fos proto-oncogene. To investigate whether there exists a relationship between mitogenic activity and c-fos expression, we analysed cellular responses (DNA synthesis and cell growth) and c-fos gene induction (mRNA and proteins) in a rat embryo fibroblast line (EL2) stimulated with epidermal growth factor (EGF), fibroblast growth factor (FGF), 12-O-tetradodecanoyl phorbol-13-acetate (TPA) and transforming growth factor beta (TGF beta). Our results suggest that the susceptibility of EL2 cells to a growth factor could be predicted as a function of the c-fos expression caused by the same growth factor. These also indicate that the c-fos gene expression may have contributed to moving our cells out of the quiescent state, but it is not the only essential event required to effect EL2 cell growth.

Proliferative response and oncogene expression induced by epidermal growth factor in EL2 rat fibroblasts.

Extensive evidence supports a two-step model for the control of fibroblast growth, which includes first the action of a competence factor (e.g., platelet-derived growth factor) followed by the stimulus of a progression factor (e.g., epidermal growth factor [EGF]). We investigated whether this model may be applied to the euploid EL2 fibroblast line recently isolated from rat embryos (E. Liboi, M. Caruso, and C. Basilico, Mol. Cell. Biol. 4:2925-2928, 1984). Our results clearly show that EGF alone leads EL2 cells to proliferate in serum-free conditions at a rate corresponding to 50 to 60% of that observed in the presence of 10% calf serum. It is of interest that, when resting EL2 cells were exposed to EGF, transcription of both c-myc and c-fos was markedly induced. Altogether, these observations suggest that, in contrast with the model of fibroblast growth mentioned above, EL2 cells require the presence of a single growth factor (EGF) for induction of DNA synthesis, and the expression of myc and fos proto-oncogenes may represent an obligatory step in the pathway of commitment of EL2 cells to proliferation. In addition, we showed that EGF may induce EL2 cells to acquire some properties of transformed cells, such as growth in agar and loss of contact inhibition. This suggests that the particular response to EGF of the EL2 line may be strictly connected with the expression of a transformed phenotype.

New rat cell line that is highly susceptible to transformation by several oncogenes.

We describe here a new cell line, EL2, which spontaneously arose from primary rat embryo fibroblasts and has the distinctive property of being highly susceptible to a number of different transforming genes. The high susceptibility is expressed not only in high transformation frequencies but, most importantly, in an unusually high rate of growth of EL2 transformants under selective conditions, i.e., in soft agar or as foci. The biological characteristics of EL2 cells greatly accelerate the isolation of transformants from known oncogenes and could be useful to detect new transforming genes.

Inhibition of polyoma gene expression in transformed mouse cells by hypermethylation.

The evolution of mouse cells transformed by a recombinant plasmid containing the genome of the tsA mutant of polyoma virus (Py) cloned at the BamHI site into the plasmid pML, whose sequences therefore interrupt the Py late region, has been studied. Clones of transformed cells were selected at 39 degrees (nonpermissive temperature for large T antigen). Under these conditions viral DNA integration is stable and the cells display a uniformed transformed phenotype. Also studied in detail was the evolution of one of these cell lines (A4) upon shift to a temperature permissive for large T-Ag function (33 degrees); immediately after shift, 90% of the population became intensely positive for T-Ag and a considerable amount of free-viral DNA was produced, accompanied by a clear cytopathic effect. Surviving cells proliferated actively after 4 weeks at 33 degrees and showed a decreased expression of large T-Ag (only 2-3% of the population was T-Ag positive by immunofluorescence), a drastic reduction in the amount of free-viral DNA produced, but no apparent change in the pattern of integration of Py DNA in the host chromosomes. Analysis of the high-molecular-weight DNA with the restriction enzymes HpaII and MspI revealed that the cytosines in the recognition sequences of these enzymes were methylated. Accordingly, treating the cells with 5-Azacytidine, a methylation inhibitor, results in the expression of viral T-Ags in more than 80% of the cell population. Analysis of DNA transcription revealed a dramatic reduction of virus-specific poly(A)+ mRNA in the methylated cells; in addition, the phenotype of the 33 degrees A4 populations was much less transformed than that of the original cultures. The block of Py expression by methylation is not complete; approximately 2% of the cells remain T-Ag positive and viral transcription is not completely suppressed. This could be explained by an incomplete methylation which randomly leaves unmethylated sequences essential for Py gene expression, or by the fact that methylation is not sufficient to block transcription completely. Possible mechanisms underlying this type of evolution are discussed.

Ultrastructural and biochemical evidence that the L929 cell retrovirus lacks the env gene translation product.

LCV, a murine retrovirus released by L929 mouse cell fibroblasts, is non-infectious when inoculated into SC-1, mink, D-17 or Vero cells. Ultrastructural examination by thin sectioning, freeze-etching or negative staining revealed the absence, on the viral envelope, of the radially disposed spikes. Polyacrylamide gel electrophoresis of radiolabelled viral components showed the absence of the glycosylated protein gp70 as well as of the p15E cleavage product of the polyprotein precursor gPr90env. The premature loss of the gp70 molecule from LCV to the culture medium was ruled out since no peak of D-[14C]glucosamine-labelled glycoprotein was detected by affinity chromatography or immunoprecipitation of concentrated medium. The ultrastructural and biochemical results all supported the hypothesis that the absence of infectivity was due to the lack of gp70 glycoprotein in the envelope of LCV. A possible block at a translational or post-translational level was also investigated by immunofluorescence studies with antisera directed against ecotropic or xenotropic gp70; Moloney murine leukaemia virus-infected or NZB cells were used as positive controls for eco- or xenotropic viruses respectively. The absence of fluorescent stain in L929 cells further supported these results and suggested that LCV and the L929 parental cell line lack the uncleaved precursor and the final product of the env gene translation process.

Identification by monoclonal antibodies of a new epitope in the glycoprotein complex of Sindbis virus.

Monoclonal antibodies against a deletion mutant of Sindbis virus were produced and characterized in order to determine the fine mapping and functional activities of single viral epitopes. All monoclonal antibodies so far tested showed a certain degree of reciprocal competition and were directed against an antigenic determinant which was present only on the undissociated complex of the E1 and E2 glycoproteins. A biological assay measuring viral haemagglutination showed no decrease in the titre of viral samples preincubated with monoclonal antibodies. Conversely, a reduction in viral infectivity was demonstrated, particularly with two of these antibodies. The results suggest that the antibodies which we characterized seem to recognize a new epitope which is represented on both glycoproteins on the surface of this mutant of Sindbis virus.