Marcio Chedid

Keratinocyte growth factor

Keratinocyte growth factor (KGF) is a member of the heparin‐binding fibroblast growth factor family (FGF‐7) with a distinctive pattern of target‐cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal‐epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr‐2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex sterorid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.

Mechanisms of Hepatocyte Growth Factor Stimulation of Keratinocyte Metalloproteinase Production

Matrix metalloproteinases participate in normal physiologic processes; however, their overproduction has been associated with connective tissue destruction in a variety of pathological states. Migrating basal keratinocytes transiently express collagenase-1 during normal cutaneous reepithelialization. However, the overexpression of both collagenase-1 and stromelysin-1 has been associated with the pathogenesis of chronic nonhealing ulcers. Aberrant expression of metalloproteinases in inflammation is mediated, at least in part, by soluble factors. Since hepatocyte growth factor/scatter factor (HGF/SF) has been reported to promote keratinocyte migration and proliferation, key events in wound repair, and since HGF/SF is produced by dermal fibroblasts and its c-Met receptor is expressed by basal keratinocytes in wounded skin, we have studied the effects of HGF/SF upon keratinocyte metalloproteinase expression. We have found that HGF/SF can stimulate keratinocyte collagenase-1 and stromelysin-1 production in a dose-dependent and matrix-dependent manner. Expression of 92-kDa gelatinase was not affected by HGF/SF. We determined that HGF/SF regulation of collagenase-1 expression is transcriptionally mediated and requires tyrosine kinase and protein kinase C activaties. HGF/NK1, a naturally occurring, truncated form of HGF/SF, also stimulates collagenase-1 production, but much less efficiently than does the parent molecule. However, HGF/NK2, another HGF/SF splice variant, as well as heparin, potently inhibit HGF/SF-induced collagenase-1 synthesis. These results indicate that HGF/SF and its naturally occurring splice variants have diverse biological effects on keratinocytes and suggest an additional mechanism whereby HGF/SF may regulate keratinocyte function during wound repair.

Keratinocyte growth factor as a cytokine that mediates mesenchymal-epithelial interaction

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, though from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and analysis of agents regulating KGF expression reinforced the idea that KGF acts predominantly on epithelial cells. While the data do not implicate a KGF autocrine loop in neoplasia, paracrine sources of factor or ligand-independent signaling by the KGFR might contribute to malignancy. Alternatively, because of its differentiation-promoting effects, KGF may retard processes that culminate in uncontrolled cell growth.

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.

DIFFERENTIAL EXPRESSION OF THE KERATINOCYTE GROWTH FACTOR (KGF) AND KGF RECEPTOR GENES IN HUMAN VASCULAR SMOOTH MUSCLE CELLS AND ARTERIES

Keratinocyte growth factor (KGF) is a secreted member of the fibroblast growth factor (FGF) family of heparin‐binding proteins. Studies reported to date indicate that it functions primarily as an important paracrine mediator of epithelial cell growth and differentiation. KGF appears to act via binding to a specific FGF receptor‐2 isoform generated by an alternative splicing mechanism. To determine whether KGF may play a role in vascular smooth muscle cell (SMC) biology, we investigated KGF and KGF receptor gene expression in human SMC cultured in vitro as well as in several human nonatherosclerotic artery and atheroma specimens. KGF mRNA but not KGF receptor mRNA was expressed by SMCs, as determined by Northern blot hybridization analysis or reverse transcription‐polymerase chain reaction assays, respectively. Additional experiments demonstrated that (1) human SMCs produce and secrete mitogenically active KGF and that (2) the cytokine interleukin‐1 increases KGF mRNA and protein levels in human SMCs. We also found that KGF transcripts but not KGF receptor transcripts were expressed in control and atherosclerotic human arteries. Taken together, these results indicate that KGF is unlikely to be involved in SMC growth regulation unless it can function intracellularly or interact with a presently unidentified KGF receptor. J. Cell. Physiol. 173:380–386, 1997. Published 1997 Wiley‐Liss, Inc. This article was prepared by a group of United States government employees and non‐United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

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.

Cloning and characterization of the mouse homolog of the human A6 gene.

The mouse homolog of a novel human protein tyrosine kinase encoding gene, A6, was cloned and characterized. The human A6 cDNA is unique in that its gene product exhibited in vitro kinase activity but its predicted amino acid (aa) sequence revealed no consensus motifs commonly found within the kinase domain of protein kinase family members. Here, we isolated a mouse A6 cDNA clone from a murine myeloid progenitor 32D cell library using a 1.1 kb cDNA probe containing the entire human A6 open reading frame (ORF). Determination of the mouse A6 cDNA nucleotide (nt) sequence revealed an ORF of 1050 nt encoding a protein of 350 aa and a molecular mass of 40,201 Da. The mouse and human A6 gene products shared 93% identity. In vitro translation, as well as immunoprecipitation of 32D cell lysates confirmed expression of mouse A6 as a 40 kDa protein. Northern blot analysis of total RNA from mouse cell lines derived from diverse tissues including NIH 3T3 fibroblasts, L cell fibroblasts, C2C12 myoblasts, M1 myeloblasts, BALB/MK cells, 70Z/3 preB lymphocytes, and p388D1 monocytes demonstrated widespread A6 mRNA expression. A6 mRNA was also ubiquitously expressed at varying levels in all tissues examined. The identification of a potential actin/phosphoinositide binding domain and consensus phosphorylation sites, coupled with A6s expression in a variety of cell types suggest that the A6 gene product may play a role in basic cellular processes.

An unexpected transforming gene in calf-thymus carrier DNA: bovine hst

During a search for transforming genes by transfecting a human cDNA expression library together with calf thymus carrier-DNA into NIH/3T3 cells, we found a focus which was induced by a plasmid containing a sequence highly homologous to human HST (a transforming gene from Human STomach cancer). However, PCR analysis identified the source of this sequence as calf thymus DNA. The deduced amino acid (aa) sequence of bovine HST shows 91 and 81% identity to the human and mouse HST aa sequences, respectively. These data suggest that the hst of calf thymus carrier-DNA could induce transformation of NIH/3T3 cells.