Effects of hepatocyte growth factor (HGF) and activin a on the morphogenesis of rat submandibular gland-derived epithelial cells in serum-free collagen gel culture

Summary To study the mechanisms of morphogenesis in salivary gland regeneration, we have established the RSMG-1 cell line derived from submandibular gland (SMG) of 10-wk-old Wistar female rats in serum-free culture. Our finding that RSMG-1 cells originated from duct cells was based on morphology and immunohistochemical results. In three-dimensional serum-free collagen gel culture, HGF induced branching morphogenesis of RSMG-1 cells. Histological examination revealed that HGF-induced branching structure exhibited well-formed lumina. This morphology closely resembles that found in vivo. The cells also expressed activin A. Exogenously added activin A at a high concentration reduced HGF-induced branching morphogenesis. These findings suggest that the morphogenesis of the salivary gland is modulated by HGF and activin A. Our results show that the RSMG-1 cell line may be useful in studies of salivary gland regeneration.     

Heterogeneity in E. coli RecBCD Helicase-DNA Binding and Base Pair Melting

E. coli RecBCD, a helicase/nuclease involved in double stranded (ds) DNA break repair, binds to a dsDNA end and melts out several DNA base pairs (bp) using only its binding free energy. We examined RecBCD-DNA initiation complexes using thermodynamic and structural approaches. Measurements of enthalpy changes for RecBCD binding to DNA ends possessing pre-melted ssDNA tails of increasing length suggest that RecBCD interacts with ssDNA as long as 17–18 nucleotides and can melt at least 10–11 bp upon binding a blunt DNA end. Cryo-EM structures of RecBCD alone and in complex with a blunt-ended dsDNA show significant conformational heterogeneities associated with the RecB nuclease domain (RecB^Nuc) and the RecD subunit. In the absence of DNA, 56% of RecBCD molecules show no density for the RecB nuclease domain, RecB^Nuc, and all RecBCD molecules show only partial density for RecD. DNA binding reduces these conformational heterogeneities, with 63% of the molecules showing density for both RecD and RecB^Nuc. This suggests that the RecB^Nuc domain is dynamic and influenced by DNA binding. The major RecBCD-DNA structural class in which RecB^Nuc is docked onto RecC shows melting of at least 11 bp from a blunt DNA end, much larger than previously observed. A second structural class in which RecB^Nuc is not docked shows only four bp melted suggesting that RecBCD complexes transition between states with different extents of DNA melting and that the extent of melting regulates initiation of helicase activity.     

Comparative phenotypic analyses of human plasma and urinary retinol binding protein using mass spectrometric immunoassay

Hepatocyte growth factor (HGF) is a mesenchymal-derived cytokine. It exerts in vitro a motogenic effect on various target cells, which is displayed either by cell scattering, locomotion, and migration during the wound repair process of cultured cells, or invasiveness through the extracellular matrix. Although it is known that HGF influences the motogenic effect of endothelial cells, the precise effects of HGF during angiogenesis are still poorly understood. To identify genes regulated via HGF signaling in HUVECs, we used the differential display polymerase chain reaction. In this study, thymosin beta4 was found to be differentially expressed in HGF-treated HUVECs compared with control. Data from HPLC profile and induction of MMPs indicate that HGF may affect the biological behavior of HUVECs through a combination of the direct effects of HGF itself and indirect effects mediated via induction of thymosin beta4 in vitro.     

Two Alternative Conformations of a Voltage-Gated Sodium Channel

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9 Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4–S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.     

Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8

It is known that chemokine-like factor superfamily 8 (CKLFSF8), a member of the CKLF superfamily, has four putative transmembrane regions and a MARVEL domain. Its structure is similar to TM4SF11 (plasmolipin) and widely distributed in normal tissue. However, its function is not yet known. We show here that CKLFSF8 is associated with the epidermal growth factor receptor (EGFR) and that ectopic expression of CKLFSF8 in several cell lines suppresses EGF-induced cell proliferation, whereas knockdown of CKLFSF8 by siRNA promotes cell proliferation. In cells overexpressing CKLFSF8, the initial activation of EGFR was not affected, but subsequent desensitization of EGF-induced signaling occurred rapidly. This attenuation was correlated with an increased rate of receptor endocytosis. In contrast, knockdown of CKLFSF8 by siCKLFSF8 delayed EGFR endocytosis. These results identify CKLFSF8 as a novel regulator of EGF-induced signaling and indicate that the association of EGFR with four transmembrane proteins is critical for EGFR desensitization.     

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N‐linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence‐activated cell sorting (FACS) and site‐specific gene excision to establish high‐yield glycoprotein expression for structural studies with stable clones derived from the well‐established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single‐chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome‐associated membrane protein 3 (LAMP3d). In both cases, stable GFP‐expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.     

Green tea (-)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.