Villars, F., B. Guillotin, T. Amé dé e, S. Dutoya, L. Bordenave, R. Bareille, and J. Amé dé e. Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication. Am J Physiol Cell Physiol 282: C775-C785, 2002; 10.1152/ajpcell.00310.2001.-Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Our aim was to investigate the interaction between human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC). Cell differentiation analysis performed with different cell culture models revealed that alkaline phosphatase activity and type I collagen synthesis were increased only by the direct contact of HUVEC with HBMSC. This "juxtacrine signaling" could involve a number of different heterotypic connexions that require adhesion molecules or gap junctions. A dye coupling assay with Lucifer yellow demonstrated a functional coupling between HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSC but also in the endothelial cell network and that these two cell types can communicate via a gap junctional channel constituted at least by Cx43. Moreover, functional inhibition of the gap junction by 18␣-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVEC cocultures on HBMSC differentiation. This stimulation could be mediated by the intercellular diffusion of signaling molecules that permeate the junctional channel.coupling; intercellular messenger; connexin43; osteoblast; endothelial cells ANGIOGENESIS is a tightly regulated process involved in growth, repair, and bone remodeling (9, 11-13, 23, 28, 50, 54, 56). Several studies have shown that there is a reciprocal regulation and functional relationship between endothelial cells and osteoblast-like cells during osteogenesis, in which systemic hormones and paracrine growth factors or cytokines play an active role (4,24,53,55). In a previous study (51), we showed that osteoblast progenitor cells [human bone marrow stromal cells (HBMSC)] behave differently in terms of proliferation and differentiation when cultured in association with endothelial cells [human umbilical vein endothelial cells (HUVEC)] in various coculture models (coculture with or without direct contact, conditioned medium) than when they are cultured alone. An increase in alkaline phosphatase activity (Al-P) was observed only when HBMSC were cocultured in direct contact with HUVEC. The enhancement of this early osteoblastic marker is not provided when HBMSC and HUVEC are cocultured separately with the use of a semipermeable membrane (51) or when HBMSC are seeded onto a matrix obtained from HUVEC cultures (51).Therefore, the intercommunication between endothelial cells and os...
Vacuolar proton pyrophosphatases (V-H؉ -PPases) are electrogenic proton pumps found in many organisms of considerable industrial, environmental, and clinical importance. V-H ؉ -PPases of several parasites were shown to be associated with acidic vacuoles named acidocalcisomes, which contain polyphosphate and calcium. In this work we functionally characterized a Trypanosoma brucei V-H ؉ -PPase gene by using double-stranded RNA interference methodology to produce inducible V-H ؉ -PPase-deficient strains of procyclic and bloodstream forms (PFiVP1 and BFiVP1). Acidocalcisomes of these mutated parasites lost acidity and contained 90% less polyphosphate. PFiVP1 did not release calcium after the addition of nigericin, and its total acidity was reduced by 70%. This mutant also failed to stabilize its intracellular pH on exposure to external basic pH >7.4 and recovered from intracellular acidification at a slower rate and to a more acidic final intracellular pH. In the absence of T. brucei V-H ؉ -PPase expression, PFiVP1 and BFiVP1 grew at a slower rate with doubling times of 27 h instead of 15 h, and 10 h instead of 7.5 h, respectively. Moreover, BFiVP1 could not grow over 5 ؋ 10 5 cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.Intracellular acidic vacuoles containing polyphosphate (polyP), 1 initially called volutin or polyP bodies, have been described in bacteria, algae, yeast, and protozoa (1). In trypanosomatids, these polyP vacuoles were called acidocalcisomes (2) and shown to be electron dense and contain large concentrations of PP i , calcium, magnesium, and other elements (3). Similar organelles have been identified in apicomplexan parasites (4, 5) as well as in the green algae, Chlamydomonas reinhardtii (6) and the slime mold, Dictyostelium discoideum (7). Acidocalcisomes were postulated to play an important role in the regulation of both cytosolic Ca 2ϩ concentration and intracellular pH (pH i ). For example, polyP hydrolysis (8) and activation of the Na ϩ /H ϩ antiporter (9, 10) were postulated to protect the cells against alkaline pH stress and increase the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ). In addition, these organelles possess a vacuolar-type H ϩ -translocating pyrophosphatase (V-H ϩ -PPase) (11), which is an electrogenic proton pump initially discovered in photosynthetic bacteria and plants (12). It has been shown to be associated with the plasma membrane or vacuoles in plants and with the chromatophore membranes of Rhodospirullum rubrum. The biochemical function of this enzyme in plants and unicellular eukaryotes is to couple hydrolysis of the high energy phosphate bond of PP i with H ϩ translocation from the cytosol to acidify the plant vacuole (tonoplast) or the acidocalcisome, respectively (10, 13). Working on isolated acidocalcisomes, Rodrigues et al. (14) demonstrated that the Trypanosoma brucei H ϩ -PPase was able to generate a membrane potential by PP i -dependent proton uptake. Moreover, Scott et al. (11,15) showed tha...
Kinesins are cytoskeletal motor proteins that play roles in a variety of fundamental cellular processes including cell division and the anterograde transport of vesicles and organelles. We purified, cloned, and functionally characterized in Trypanosoma brucei a new member of the C-terminal kinesin family, TbKIFC1. Kinetic constants of the recombinant motor domain of TbKIFC1 were estimated at 0.56 M for the microtubule dissociation constant (K d ) with a k cat of 0.2 s ؊1 . Immunolocalization analysis showed an association of TbKIFC1 with punctate structures. Because they were rapidly transported to the negative pole of the microtubule after NH 4 Cl treatment, these structures were considered to be associated with acidic vesicles. To determine the role of the kinesin in vivo, we produced an inducible kinesin-deficient strain by double-stranded RNA interference methodology.
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