BBA - Molecular Cell Research (v.1746, #1)

It is well known that the biological and carcinogenic effects of 17β-estradiol (E2) are mediated via nuclear estrogen receptors (ERs) by regulating nuclear gene expression. Several rapid, non-nuclear genomic effects of E2 are mediated via plasma membrane-bound ERs. In addition, there is accumulating evidence suggesting that mitochondria are also important targets for the action of estrogens and ERs. This review summarized the studies on the effects of estrogens via ERs on mitochondrial structure and function. The potential physiological and pathophysiological implications of deficiency and/or overabundance of these E2/ER-mediated mitochondrial effects in stimulation of cell proliferation, inhibition of apoptosis, E2-mediated cardiovascular and neuroprotective effects in target cells are also discussed.
Keywords: 17β-estradiol; Estrogen; Estrogen carcinogenesis; Estrogen receptors α and β; Mitochondria; Mitochondrial DNA-encoded gene; Mitochondrial DNA transcription; Mitochondrial estrogen receptor; Mitochondrial respiratory chain (MRC); Nuclear genes for MRC protein;

Evaluation of rage isoforms, ligands, and signaling in the brain by Qunxing Ding; Jeffrey N. Keller (18-27).
Since the identification of the receptor for advanced glycosylation end products (RAGE) in 1992, there have been tremendous strides made in our understanding of the role RAGE receptors play in a variety of physiological and pathological processes. Despite such progress, several fundamental aspects of RAGE expression and RAGE function remain largely unanswered. In particular, while multiple forms of the RAGE receptor are known to exist, little is known with regards to how these different isoforms of the RAGE receptor work together to mediate RAGE signaling. For example, some forms of the RAGE receptor may promote deleterious feed-forward pathways, while others may serve to inhibit deleterious activation of the RAGE receptor. Additionally, important questions remain with regards to the intracellular domain of the full-length RAGE receptor, and the specifics surrounding how intracellular signaling pathways become activated via the RAGE family of receptors. The focus of this review is to address each of these important issues, as well as other key aspects of RAGE biology, and discuss how they are important for both our understanding of the physiological and pathological roles of RAGE signaling within the brain.
Keywords: Advanced glycosylation end product; Alzheimer's disease; Amphoterin; Beta amyloid; HMG-1; Neurodegeneration; Oxidative stress;

RGDS and DGEA-induced [Ca2+]i signalling in human dermal fibroblasts by P. Mineur; A. Guignandon; Ch.A. Lambert; M. Amblard; Ch.M. Lapière; B.V. Nusgens (28-37).
A pulse of short peptides, RGDS and DGEA in the millimolar range, immediately elicits in normal human fibroblasts a transient increase of intracellular Ca2+ ([Ca2+]i). In the present study, we show that this [Ca2+]i occurs in an increasing number of cells as a function of peptides concentration. It is specific of each peptide and inhibited at saturating concentration of the peptide in the culture medium. The [Ca2+]i transient depends on signalling pathways slightly different for DGEA and RGDS involving tyrosine kinase(s) and phosphatase(s), phospholipase C, production of inositol-trisphosphate and release of Ca2+ from the cellular stores. GFOGER, the classical collagen binding peptide of α1- α2- and α11-β1 integrins, in triple helical or denatured form, does not produce any Ca2+ signal. The [Ca2+]i signalling induced by RGDS and DGEA is inhibited by antibodies against β1 integrin subunit while that mediated by RGDS is also inhibited by antibodies against the α3 integrin. Delay in the acquisition of responsiveness is observed during cell adhesion and spreading on a coat of fibronectin for RGDS or collagen for DGEA or on a coat of the specific integrin-inhibiting antibodies but not by seeding cells on GFOGER or laminin-5. This delay is suppressed specifically by collagenase acting on the collagen coat or trypsin on the fibronectin coat. Our results suggest that free integrins and associated focal complexes generate a Ca2+ signal upon recognition of DGEA and RGDS by different cellular pathways.
Keywords: Integrin; Ca2+ signalling; RGDS; DGEA; Fibroblast;

Earlier studies from this laboratory showed that human folylpolyglutamate synthetase (FPGS) exists as cytosolic and mitochondrial (mFPGS) isoforms. Localization of mFPGS within mitochondria may help elucidate how the enzyme functions to maintain the mitochondrial folate pool. A human T-lymphoblastic leukemia CCRF-CEM cell lysate was fractionated by differential centrifugation into cytosolic and mitochondrial fractions. Activity assays for cytosol-and mitochondria-specific enzymes verified the purity and integrity of the fractions. Mitochondria were subfractionated with increasing concentrations of digitonin to successively extract the four submitochondrial compartments. Western analyses of the fractions using protein markers specific for each compartment suggest that mFPGS is distributed in the matrix and/or inner membrane compartments. Further support for an interaction of mFPGS with the inner mitochondrial membrane is provided by localization of about half of the mFPGS in the mitochondrial membrane fraction obtained by freeze–thaw of intact mitochondria; the remaining mFPGS is located in the soluble fraction. Resistance of about half of the mFPGS in whole mitochondria to alkaline carbonate extraction suggests that its interaction with the inner membrane is more similar to an integral, than a peripheral, membrane protein. The data suggest that human mFPGS is at least in part strongly associated with the inner mitochondrial membrane.
Keywords: Folylpolyglutamate synthetase; Polyglutamate; Mitochondria; Folate; Leukemia; CCRF-CEM;

CaMK-II oligomerization potential determined using CFP/YFP FRET by Konstantin Lantsman; Robert M. Tombes (45-54).
Members of the Ca2+/calmodulin-dependent protein kinase II (CaMK-II) family are encoded throughout the animal kingdom by up to four genes (α, β, γ, and δ). Over three dozen known CaMK-II splice variants assemble into ∼12-subunit oligomers with catalytic domains facing out from a central core. In this study, the catalytic domain of α, β, and δ CaMK-IIs was replaced with cyan (CFP) or yellow fluorescent protein (YFP) for fluorescence resonance energy transfer (FRET) studies. FRET, when normalized to total CFP and YFP, reproducibly yielded values which reflected oligomerization preference, inter-subunit spacing, and localization. FRET occurred when individual CFP and YFP-linked CaMK-IIs were co-expressed, but not when they were expressed separately and then mixed. All hetero-oligomers exhibited FRET values that were averages of their homo-oligomeric parents, indicating no oligomeric preference or restriction. FRET for CaMK-II homo-oligomers was inversely proportional to the variable region length. FPs were monomerized (Leu221 to Lys221) for this study, thus eliminating any potential artifact caused by FP-CaMK-II aggregates. Our results indicate that α, β, and δ CaMK-IIs can freely hetero-oligomerize and that increased variable region lengths place amino termini further apart, potentially influencing the rate of inter-subunit autophosphorylation.
Keywords: CaM kinase II; FRET; CFP; YFP; Localization; Oligomerization;

Integrin α1β1 mediates collagen induction of MMP-13 expression in MC615 chondrocytes by Marie-Claire Ronzière; Elisabeth Aubert-Foucher; Jérôme Gouttenoire; Janine Bernaud; Daniel Herbage; Frédéric Mallein-Gerin (55-64).
During endochondral ossification, type I collagen is synthesized by osteoblasts together with some hypertrophic chondrocytes. Type I collagen has also been reported to be progressively synthesized in degenerative joints. Because Matrix Metalloproteinase-13 (MMP-13) plays an active role in remodeling cartilage in fetal development and osteoarthritic cartilage, we investigated whether type I collagen could activate MMP-13 expression in chondrocytes. We used a well-established chondrocytic cell line (MC615) and we found that MMP-13 expression was induced in MC615 cells cultured in type I collagen gel. We also found that α1β1 integrin, a major collagen receptor, was expressed by MC615 cells and we further assessed the role of α1β1 integrin in conducting MMP-13 expression. Induction of MMP-13 expression by collagen was potently and synergistically inhibited by blocking antibodies against α1 and β1 integrin subunits, indicating that α1β1 integrin mediates the MMP-13-inducing cellular signal generated by three-dimensional type I collagen. We also determined that activities of tyrosine kinase and ERK and JNK MAP kinases were required for this collagen-induced MMP-13 expression. Interestingly, bone morphogenetic protein (BMP)-2 opposed this induction, an effect that may be related to a role of BMP-2 in the maintenance of cartilage matrix.
Keywords: Chondrocyte; Collagen; Integrin; MMP-13; BMP-2; Osteoarthritis;

Two-hybrid analysis of human salivary mucin MUC7 interactions by Lucila S. Bruno; Xiaojing Li; Li Wang; Rodrigo V. Soares; Camille C. Siqueira; Frank G. Oppenheim; Robert F. Troxler; Gwynneth D. Offner (65-72).
MUC7 is a low molecular weight monomeric mucin secreted by submandibular, sublingual and minor salivary glands. This mucin has been implicated in the non-immune host defense system in the oral cavity since it binds and agglutinates a variety of oral microbes. To investigate interactions between this mucin and other secretory salivary proteins, a submandibular gland prey library was screened with baits encoding the N- and C-terminal regions of MUC7 in the yeast two-hybrid system. The N-terminal region interacted with several secretory salivary proteins, whereas the C-terminal region did not. Interacting proteins included amylase, acidic proline-rich protein 2, basic proline-rich protein 3, lacrimal proline-rich protein 4, statherin and histatin 1. Formation of complexes between these proteins and the N-terminal region of MUC7 was confirmed in Far Western blotting experiments. Interactions between mucin and non-mucin proteins in saliva could protect complex partners from proteolysis, modulate the biological activity of complexed proteins or serve as a delivery system for distribution of secretory salivary proteins throughout the oral cavity.
Keywords: Mucin; Protein–protein interaction; MG2; Salivary protein;