Matthew V. Lorenzi

Identification and characterization of R-ras3 : a novel member of the RAS gene family with a non-ubiquitous pattern of tissue distribution

Members of the Ras subfamily of GTP-binding proteins, including Ras (H-, K-, and N-), TC21, and R-ras have been shown to display transforming activity, and activating lesions have been detected in human tumors. We have identified an additional member of the Ras gene family which shows significant sequence similarity to the human TC21 gene. This novel human ras-related gene, R-ras3, encodes for a protein of 209 amino acids, and shows ∼60 – 75% sequence identity in the N-terminal catalytic domain with members of the Ras subfamily of GTP-binding proteins. An activating mutation corresponding to the leucine 61 oncogenic lesion of the ras oncogenes when introduced into R-ras3, activates its transforming potential. R-ras3 weakly stimulates the mitogen-activated protein kinase (MAPK) activity, but this effect is greatly potentiated by the co-expression of c-raf-1. By the yeast two-hybrid system, R-ras3 interacts only weakly with known Ras effectors, such as Raf and RalGDS, but not with RglII. In addition, R-ras3 displays modest stimulatory effects on trans-activation from different nuclear response elements which bind transcription factors, such as SRF, ETS/TCF, Jun/Fos, and NF-κB/Rel. Interestingly, Northern blot analysis of total RNA isolated from various tissues revealed that the 3.8 kilobasepair (kb) transcript of R-ras3 is highly restricted to the brain and heart. The close evolutionary conservation between R-ras3 and Ras family members, in contrast to the significant differences in its biological activities and the pattern of tissue expression, raise the possibility that R-ras3 may control novel cellular functions previously not described for other GTP-binding proteins.

Tumor necrosis factor-α induces the expression of DR6, a member of the TNF receptor family, through activation of NF-κB

The tumor necrosis factor (TNF) receptor family are ligand-regulated transmembrane proteins that mediate apoptosis as well as activation of the transcription factor NF-κB. Exogenous expression of DR6, a recently identified member of the TNF receptor family, induced apoptosis in untransformed or tumor-derived cells and the apoptotic function of DR6 was inhibited by co-expression of Bcl-2, Bcl-xL or the inhibitor-of-apoptosis (IAP) family member, survivin. Expression of a dominant negative mutant of FADD failed to protect from DR6-mediated apoptosis indicating that unlike TNFR1 and Fas, DR6 induced apoptosis via a FADD-independent mechanism. Despite the ability of exogenous DR6 expression to induce apoptosis, DR6 mRNA and protein were found to be elevated in prostate tumor cell lines and in advanced stages of prostate cancer. Analysis of several anti-apoptotic proteins revealed that Bcl-xL levels and serine 32 phosphorylation of IκB, the natural inhibitor of NF-κB, were similarly elevated in cells expressing high levels of DR6, suggesting that NF-κB-regulated survival proteins may protect from DR6-induced apoptosis and that DR6 is a target of NF-κB regulation. Treatment of LnCAP cells with TNF-α resulted in increases in both DR6 mRNA and protein levels, and this induction was suppressed by inhibitors of NF-κB. Similarly, treatment of cells expressing high levels of DR6 with indomethacin and ibuprofen, compounds also known to perturb NF-κB function, resulted in a dose-dependent decrease in DR6 protein and mRNA levels. These results demonstrate that TNF-α signaling induces the expression of a member of its own receptor family through activation of NF-κB.

Rho exchange factor ECT2 is induced by growth factors and regulates cytokinesis through the N-terminal cell cycle regulator-related domains†

The ECT2 protooncogene plays a critical role in cytokinesis, and its C‐terminal half encodes a Dbl homology‐pleckstrin homology module, which catalyzes guanine nucleotide exchange on the Rho family of small GTPases. The N‐terminal half of ECT2 (ECT2‐N) contains domains related to the cell cycle regulator/checkpoint control proteins including human XRCC1, budding yeast CLB6, and fission yeast Cut5. The Cut5‐related domain consists of two BRCT repeats, which are widespread to repair/checkpoint control proteins. ECT2 is ubiquitously expressed in various tissues and cell lines, but elevated levels of ECT2 expression were found in various tumor cell lines and rapidly developing tissues in mouse embryos. Consistent with these findings, induction of ECT2 expression was observed upon stimulation by serum or various growth factors. In contrast to other oncogenes whose expression is induced early in G1, ECT2 expression was induced later, coinciding with the initiation of DNA synthesis. To test the role of the cell cycle regulator/checkpoint control protein‐related domains of ECT2 in cytokinesis, we expressed various ECT2 derivatives in U2OS cells, and analyzed their DNA content by flow cytometry. Expression of the N‐terminal half of ECT2, which lacks the catalytic domain, generated cells with more than 4N DNA content, suggesting that cytokinesis was inhibited in these cells. Interestingly, ECT2‐N lacking the nuclear localization signals inhibited cytokinesis more strongly than the derivatives containing these signals. Mutational analyses revealed that the XRCC1, CLB6, and BRCT domains in ECT2‐N are all essential for the cytokinesis inhibition by ECT2‐N. These results suggest that the XRCC1, CLB6, and BRCT domains of ECT2 play a critical role in regulating cytokinesis. Published 2003 Wiley‐Liss, Inc.

Neu differentiation factor/heregulin induction by hepatocyte and keratinocyte growth factors

Hepatocyte growth-factor (HGF) is a potent, widely produced, pleiotropic mediator of mesenchymal-epithelial interaction. In a study of changes in gene expression initiated by HGF in Balb/MK keratinocytes, we observed the induction of Neu-differentiation factor (NDF) mRNA (also known as heregulin, or HRG). Further characterization of the regulation of NDF expression in Balb/MK keratinocytes revealed potent induction by keratinocyte growth factor (KGF) and epidermal growth factor (EGF), but not by HGF/NK2, an alternative HGF isoform with motogenic but not mitogenic or morphogenic activities. Sustained treatment (8 h) of Balb/MK cells with KGF stimulated secretion of mature NDF protein into the culture medium, and Balb/MK cells treated with purified recombinant NDF protein showed increased DNA synthesis. We also found evidence of NDF induction in two models of tissue repair in mice: in full-thickness skin wounds, following locally increased KGF production, and in kidney after partial hepatectomy, following elevation of circulating HGF levels. These results reveal that mesenchymally-derived HGF and KGF can activate autocrine NDF signaling in their epithelial targets, and suggest that this mechanism contributes to the coordination of stages of wound repair, and possibly development, where these growth factors act in concert to direct epithelial proliferation, morphogenesis and differentiation.

Homocysteine exerts cell type-specific inhibition of AP-1 transcription factor.

Homocysteine (Hcy) exerts either promoting or suppressive effects on mitogenesis in a cell type-specific manner. Hcy elicits proliferation of vascular smooth muscle cells, but is rather inhibitory to growth of endothelial cells and NIH/3T3 cells. In NIH/3T3 cells, we found that physiologically relevant concentrations (20-100 microM) of Hcy inhibit the activity of activating protein-1 (AP-1) transcription factor, although it is capable of eliciting immediate-early signaling events. Hcy induced p44/42 mitogen-activated protein kinase (MAPK) phosphorylation in control cells, but not in dominant negative p21ras transfected cells, indicating induction of the Ras-MAPK pathway. Hcy also induced the activity of serum response factor and expression of c-fos and c-jun genes. Despite the activation of these upstream events, Hcy potently inhibited AP-1 activity. Oxidized forms of Hcy (Hcy thiolactone, homocystine) were less effective in affecting AP-1. Hcy-mediated inhibition of AP-1 activity was not observed in A7r5 vascular smooth muscle cells. These results demonstrate that Hcy exerts cell type- and redox-specific inhibition of AP-1 dependent biological events.

Distinct expression patterns and transforming properties of multiple isoforms of Ost, an exchange factor for RhoA and Cdc42.

A search for transforming genes expressed in brain led to the identification of a novel isoform of Ost, an exchange factor for RhoA and Cdc42. In addition to the Dbl-homology (DH) and pleckstrin-homology (PH) domains identified in the original Ost, this isoform contained a SH3 domain and a novel HIV-Tat related (TR) domain. The presence or absence of these domains in Ost defined multiple isoforms of the protein. RT – PCR and in situ hybridization analysis revealed that these isoforms were generated by tissue-specific and developmentally restricted alternative splicing events. Whereas deletion of the N-terminus activated the transforming properties of Ost, the presence of the SH3 domain reduced the transforming activity of the protein. This inhibition was relieved by the presence of a TR domain, which contained a potential SH3 ligand sequence. The transforming activity of all Ost isoforms was inhibited by dominant negative forms of the Rho family proteins. Expression of Ost isoforms potently induced the formation of actin stress fibers and filopodia as well as JNK activity and AP1- and SRF-regulated transcriptional pathways. Ost transfectants also displayed elevated levels of cyclins A and D1, suggesting that the de-regulation of these cyclins is linked to Ost-mediated transformation.

Ligand-independent activation of fibroblast growth factor receptor-2 by carboxyl terminal alterations

To assess the effect(s) of the C-terminal domain on FGFR2 function, we engineered a series of mutant FGFR2 cDNAs encoding deletions in the C-terminus of the receptor and compared their growth properties in NIH3T3 fibroblasts. In contrast to FGFR2-WT, receptors with C-terminal truncations induced ligand-independent transformation of NIH3T3 cells and transfectants expressing these mutant receptors efficiently formed colonies in semisolid medium. Introduction of point mutations (Y to F) into the C-terminus of FGFR2 at positions 813, 784 or 780 revealed that these mutant receptors also displayed activities similar to that of C-terminally truncated receptors. C-terminally altered FGF receptors did not show an increase in the basal level of receptor phosphorylation compared to that of FGFR2-WT suggesting that elevated receptor phosphorylation does not underlie the transforming activity of these receptors. Interestingly, expression of transforming FGFR2 derivatives, unlike H-Ras transformed cells, did not result in the activation of the mitogen-activated protein kinases (MAPKs), p42/ERK2 and p44/ERK1, indicating that this pathway is not constitutively active in FGFR2-transformed cells. Finally, we report the overexpression of FGFR2 mRNA and protein in several human tumor cell lines suggesting activation of the receptor in these tumors.

Cooperative transformation of NIH3T3 cells by Gα12 and Rac1

The heterotrimeric G-protein, Gα12, together with the closely-related Gα13, are members of the G12 class of α-subunits important in mediating the signaling from seven transmembrane domain-spanning receptors. Recent evidence implicating both Gα12 and Gα13 in the activation of signaling pathways involving members of the RHO gene family led us to examine the role of Rac1, RhoA and Cdc42Hs in the transforming properties of Gα12. Asparagine 17 (Asn 17) dominant inhibitory mutants of Rac1, and to a lesser extent RhoA, block focus forming ability of the GTPase-deficient mutant of Gα12 (Gα12 Leu 229) in NIH3T3 cells. In turn, wild-type Gα12 cooperates well with Rac1 Val 12 but not with RhoA Leu 63 mutant in transforming NIH3T3 cells. Interestingly, the morphology of foci induced by Gα12 and RhoA mutants are strikingly similar and is distinct from those displayed by Rac1 Val 12 mutant. The fact that Gα12s ability to induce mitogenesis in NIH3T3 cells is not significantly perturbed by C3 ribosyltransferase suggested that RhoA does not play a major role in Gα12-induced mitogenic events. Activated mutant of Rac1 has previously been demonstrated to stimulate the activity of the stress-induced c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs). Transient co-transfection of Rac1 Val 12 mutant with the wild-type Gα12 in COS7 cells leads to the further activation of an exogenously expressed hemagglutinin(HA)-tagged JNK. Furthermore, the cooperation between Gα12 and Rac1 in cellular transformation is correlated with their ability to stimulate transcription from c-fos serum response element (SRE).

The Acidic Domain and First Immunoglobulin-Like Loop of Fibroblast Growth Factor Receptor 2 Modulate Downstream Signaling through Glycosaminoglycan Modification

ABSTRACT Fibroblast growth factor receptors (FGFRs) are membrane-spanning tyrosine kinases that have been implicated in a variety of biological processes including mitogenesis, cell migration, development, and differentiation. We identified a unique isoform of FGFR2 expressed as a diffuse band with an unusually large molecular mass. This receptor is modified by glycosaminoglycan at a Ser residue located immediately N terminal to the acidic box, a stretch of acidic amino acids. The acidic box and the glycosaminoglycan modification site are encoded by an alternative exon of the FGFR2 gene. The acidic box appears to play an important role in glycosaminoglycan modification, and the presence of this domain is required for modification by heparan sulfate glycosaminoglycan. Moreover, the presence of the first immunoglobulin-like domain encoded by another alternative exon abrogated the modification. The high-affinity receptor with heparan sulfate modification enhanced receptor autophosphorylation, substrate phosphorylation, and ternary complex factor-independent gene expression. It also sustained mitogen-activated protein kinase activity and increased eventual DNA synthesis, a long-term response to fibroblast growth factor stimulation, at physiological ligand concentrations. We propose a novel regulation mechanism of FGFR2 signal transduction through glycosaminoglycan modification.

Identification of a novel activated form of the keratinocyte growth factor receptor by expression cloning from parathyroid adenoma tissue.

Parathyroid adenomas are benign tumors in the parathyroid glands, whose pathogenesis is largely unknown. We utilized an expression cDNA cloning strategy to identify oncogenes activated in parathyroid adenomas. An expression cDNA library was prepared directly from a clinical sample of parathyroid adenoma tissue, transfected into NIH3T3 cells, and foci of morphologically transformed cells were isolated. Following plasmid rescue, we identified cDNAs for the keratinocyte growth factor receptor at a high frequency. Interestingly, approximately half of the clones encoded a variant receptor containing an altered C-terminus. Analysis of the transforming activity of the variant receptor revealed that the altered C-terminus up-regulated the transforming activity in a ligand-independent manner. The higher transforming activity was not accompanied by increase of dimerization or overall autophosphorylation of the receptor. However, tyrosine phosphorylation of downstream receptor substrates, including Shc isoforms and possibly FRS2, are increased in the transfectants expressing the parathyroid tumor-derived receptor. Genomic analysis showed that a previously unidentified exon was used to form the novel isoform. This alternative splicing appears to occur preferentially in parathyroid adenomas.