Faculty Bibliography

Normal breast tissue as well as most breast tumors are dependent on estrogen for growth. Breast tumors often progress to a hormone-independent state which is associated with poor prognosis. It has been proposed that activation of growth factor signaling pathways in the tumor cells may free them from hormonal control. Certain growth factors can mimic estrogen responses by activating the estrogen receptor via its phosphorylation by mitogen-activated protein (MAP) kinase. In this report, however, we show that fibroblast growth factor (FGF), despite activating MAP kinase, is growth-inhibitory for estrogen-dependent MCF-7 breast cancer cells. MCF-7 cells treated with FGFs exhibit slower growth than controls in both the presence and absence of estrogen, with a concomitant increase in the number of cells in G0/G1. Expression of a constitutively activated FGF receptor in these cells further decreases their growth rate, which is no longer influenced by FGF treatment. Activation of the FGF signaling pathway also reduces the induction of an estrogen-responsive CAT reporter plasmid by estrogen, an effect which appears to be independent of serine 118 in the estrogen receptor, a MAP kinase target site. The inhibitory effects of FGF are probably mediated through the sustained induction of the cyclin kinase inhibitor p21/WAF1/CIP1, which is upregulated at the mRNA and protein level by FGF. FGF treatment also results in the phosphorylation of STAT1. This upregulation of p21 and phosphorylation of STAT1 is not detectable in T47D breast cancer cells upon which FGF has no inhibitory effect

ARK (AXL) is the prototype of a distinctive family of receptor tyrosine kinases which contain in their extracellular domains features reminiscent of cell adhesion molecules. ARK is capable of homophilic binding, which results in a degree of receptor activation, but can also be activated by a heterophilic ligand, Gas6, a member of the family of vitamin K dependent proteins that is preferentially expressed in quiescent cells. Since a number of tissues and cell lines express both ARK and Gas6, we studied the effect of endogenous and exogenous Gas6 on the phenotype of ARK expressing cells. Here we show that constitutive expression of Gas6 in an NIH3T3 cell line that does not spontaneously express this protein does not result in cell transformation or uncontrolled growth, but protects from apoptosis induced by serum deprivation. Recombinant exogenous Gas6 was also capable of protecting cells from apoptosis at concentrations that did not result in significant induction of DNA synthesis. Activation of ARK phosphorylation and a weak but significant induction of MAP kinase activity accompanied the increased survival of cells treated with Gas6. The antiapoptotic effect of ARK signaling was confirmed by studies using fibroblasts from ARK knock-out mice, that showed that the absence of ARK resulted in higher levels of serum deprivation-induced apoptosis, that could not be rescued by the addition of Gas6. Interestingly ARK signaling protects from apoptosis induced by serum deprivation, myc overexpression, or by TNF alpha but not from u.v. irradiation or Staurosporine. These results suggest that a major function of Gas6-ARK signaling is that of increasing cell survival under conditions which do not allow cell proliferation

Octamer binding and Sox factors are thought to play important roles in development by potentiating the transcriptional activation of specific gene subsets. The proteins within these factor families are related by the presence of highly conserved DNA binding domains, the octamer binding protein POU domain or the Sox factors HMG domain. We have previously shown that fibroblast growth factor 4 (FGF-4) gene expression in embryonal carcinoma cells requires a synergistic interaction between Oct-3 and Sox2 on the FGF-4 enhancer. Sox2 and Oct-3 bind to adjacent sites within this enhancer to form a ternary protein-DNA complex (Oct-3*) whose assembly correlates with enhancer activity. We now demonstrate that increasing the distance between the octamer and Sox binding sites by base pair insertion results in a loss of enhancer function. Significantly, those enhancer 'spacing mutants' which failed to activate transcription were also compromised in their ability to form the Oct* complexes even though they could still bind both Sox2 and the octamer binding proteins, suggesting that a direct interaction between Sox2 and Oct-3 is necessary for enhancer function. Consistent with this hypothesis, Oct-3 and Sox2 can participate in a direct protein-protein interaction in vitro in the absence of DNA, and both this interaction and assembly of the ternary Oct* complexes require only the octamer protein POU and Sox2 HMG domains. Assembly of the ternary complex by these two protein domains occurs in a cooperative manner on FGF-4 enhancer DNA, and the loss of this cooperative interaction contributes to the defect in Oct-3* formation observed for the enhancer spacing mutants. These observations indicate that Oct-3* assembly results from protein-protein interactions between the domains of Sox2 and Oct-3 that mediate their binding to DNA, but it also requires a specific arrangement of the binding sites within the FGF-4 enhancer DNA. Thus, these results define one parameter that is fundamental to synergistic activation by Sox2 and Oct-3 and further emphasize the critical role of enhancer DNA sequences in the proper assembly of functional activation complexes

We have identified a novel type 2C serine-threonine phosphatase, FIN13, whose expression is induced by fibroblast growth factor 4 and serum in late G1 phase. The protein encoded by FIN13 cDNA includes N- and C-terminal domains with significant homologies to type 2C phosphatases, a domain homologous to collagen, and an acidic domain. FIN13 expression predominates in proliferating tissues. Bacterially expressed FIN13 and FIN13 expressed in mammalian cells exhibit serine-threonine phosphatase activity, which requires Mn2+ and is insensitive to inhibition by okadaic acid. FIN13 is localized in the nuclei of transiently transfected cells. Cotransfection of FIN13-expressing plasmids with a plasmid that expresses the neomycin resistance gene inhibits the growth of drug-resistant colonies in NIH 3T3, HeLa and Rat-1 cells. In transiently transfected cells, FIN13 inhibits DNA synthesis and results in the accumulation of cells in G1 and early S phases. Similarly, the induction of expression of FIN13 under the control of a tetracycline-regulated promoter in NIH 3T3 cells leads to growth inhibition, with accumulation of cells in G1 and early S phases. Thus, overexpression and/or unregulated expression of FIN13 inhibits cell cycle progression, indicating that the physiological role of this phosphatase may be that of regulating the orderly progression of cells through the mitotic cycle by dephosphorylating specific substrates which are important for cell proliferation

FGF signaling is clearly important for proper bone development, and several autosomally dominant forms of genetic bone disorders have been mapped to FGF receptors 1, 2, and 3. We have studied the biological effects of the most commonly mutated cysteine residue in FGFR-2 which is detected in individuals with Crouzon syndrome, an autosomally dominant trait which causes premature fusion of the skull bones (craniosynostosis). This Crouzon mutation replaces the cysteine at position 342 with tyrosine, thus disrupting the formation of the third immunoglobulin (Ig)-like loop in the extracellular portion of the receptor. By transfecting mutated and wild-type receptors into a variety of cell lines, we have shown that the C342Y mutation in FGFR-2 produces a receptor which is constitutively activated and capable of transforming NIH3T3 cells and preventing the differentiation of C2 myoblasts in the absence of ligand. Constitutive activation appears to result from the ability of this receptor to form stable interreceptor dimers which involve disulfide bonds between the remaining free cysteine in the mutant receptor. The altered conformation of the third Ig-like domain in the mutated receptor also results in a drastically reduced ability to bind FGF-1 or FGF-2 and in a reduced level of receptor glycosylation. Thus it appears that Crouzon syndrome results from constitutive activation of FGFR-2 and that uncontrolled FGF signaling produces alterations of intramembranous bone development and premature closing of cranial sutures

In a previous study, we demonstrated that sodium salicylate (NaSal) selectively inhibits tumor necrosis factor (TNF)-induced activation of the p42 and p44 mitogen-activated protein kinases (MAPKs) (known as extracellular signal-regulated kinases). Here we show that in normal human FS-4 fibroblasts NaSal inhibits TNF-induced activation of another member of the MAPK family, the c-Jun N-terminal kinase/stress-activated protein kinase. c-Jun N-terminal kinase activation induced by interleukin 1 or epidermal growth factor was less strongly inhibited by NaSal. Unexpectedly, treatment of FS-4 cells with NaSal alone produced a strong activation of p38 MAPK and cell death by apoptosis. NaSal-induced apoptosis was blocked by the selective p38 MAPK inhibitor SB-203580, indicating that p38 MAPK serves as a mediator of NaSal-induced apoptosis in human fibroblasts. Activation of p38 MAPK and the resulting induction of apoptosis may be important in the demonstrated antineoplastic actions of nonsteroidal anti-inflammatory drugs

Signaling through FGF receptors, which constitute a family of membrane-spanning tyrosine kinases, can stimulate cell proliferation, induce or inhibit cell differentiation and plays an important role in development. Recently, mutations in FGF receptors have been shown to be associated with a number of genetically dominant human skeletal disorders. A remarkably conserved mutation (Gly 380-->Arg) in the transmembrane region of FGFR-3 has been shown to be responsible for achondroplasia (ACH) but it was not clear whether such mutations result in loss of receptor function or constitutive activation. We have therefore made mutations in the transmembrane regions of murine FGFR-2 and FGFR-3 and studied their effect on receptor activity. We show here that the ACH mutation in FGFR-3 as well as two similar mutations in FGFR-2 result in constitutive activation of these receptors. This is manifested in their ability to become autophosphorylated in the absence of ligand in L6 cells, transforming activity on NIH3T3 fibroblasts, and the ability to inhibit myogenic differentiation in the absence of growth factor. Thus the transmembrane region of FGFR-2 and FGFR-3 plays a regulatory role in receptor function and the ACH mutation produces a dominant oversignaling receptor which is no longer regulated by FGF binding. These findings also support the newly identified role of FGF signaling as a negative regulator of bone growth

The receptor tyrosine kinase ARK (also called AXL or UFO) is the murine prototype of a small family of receptors with an extracellular domain resembling cell adhesion molecules and a conserved tyrosine kinase domain. ARK is capable of homophilic binding, as well as of binding of GAS6, a secreted member of the class of vitamin K dependent proteins whose expression is up-regulated in growth-arrested cells. To gain understanding of the physiological role of ARK signaling, we have investigated the ARK forms which are expressed by cells in culture as well as by mouse organs. We found that ARK is not only expressed as a transmembrane protein, but is also cleaved in the extracellular domain to generate a soluble ARK form of about 65 kDa, which is easily detected in conditioned media of ARK expressing cells, in serum and plasma and in mouse organs. Soluble ARK is also produced by tumor cells in vivo. The function of these molecules could be that of binding GAS6, thereby inhibiting the interaction of this ligand with its cell-associated receptor, or they could be involved in binding to ARK itself