Here, we analyzed the role of the cell adhesion molecule JAM-C, a protein known to mediate cellular polarity during spermatogenesis, in hematopoiesis. ofJam-Cleads to an increase in a5IA myeloid progenitors and granulocytes in the BM. Stem cells and myeloid cells from fetal liver are normal in number and homing to the BM. These results provide evidence that JAM-C defines a5IA HSCs in the BM and that JAM-C plays a role in controlling myeloid progenitor generation in the BM. == Introduction == There is increasing evidence that interactions of stem cells with the environment are key in maintaining the balance between self-renewal and differentiation. Adult hematopoietic stem cells (HSCs) reside in a special microenvironment known as the niche, which provides extrinsic regulatory signals that control intrinsic genetic programs required for HSC function.13A number of cell-surface molecules on HSCs have been shown to regulate the maintenance of HSCs. Among Rabbit Polyclonal to BEGIN others, these include bone morphogenic proteins,4Ca-sensing receptor,5Notch,684,9and Tie2.10In addition, transcription profiling of the most primitive HSCs has identified cell junction proteins, which were previously not implicated in stem cell functions, to be differentially expressed1113; moreover, adhesion and junction complexes have been proposed to trigger molecular signals, influencing the balance between self-renewal and differentiation.14 The junctional adhesion molecule JAM-C is a member of a family of adhesion molecules belonging to the immunoglobulin (Ig) superfamily. JAM-C was found to be expressed on a number of different cells such as endothelial1517and epithelial cells,18fibroblasts,19smooth muscle cells,20spermatids,21,22and peripheral nerves.23Expression of JAM-C on platelets24and lymphocytes16,25is restricted to human tissue. JAM-C interacts via its ectodomain homotypically26,27and heterotypically with the integrins M2and X218,24, JAM-B,16,2830and the viral receptor CAR.21Through the c-terminal PDS95/Dig/ZO-1 (PDZ) domain-binding motif, JAM-C associates with the PDZ domain containing proteins ZO-1, Par-3, Par-6, PATJ, and PICK-1,22,31,32and localizes to cell-cell junctions.17,19,20,33,34 The broad expression and variety of counterreceptors suggests that JAM-C regulates heterotypic cell-cell interactions, such as leukocyte-endothelial interactions in the immune system, as well as homotypic cell-cell interactions, such as cellular junctions in endothelial and epithelial cells.3539Blocking of JAM-C function inhibits leukocyte migration in several in vivo models of inflammation,18,25,34,4043leukocyte-platelet interactions,24,44and neovascularization in models of angiogenesis, a process that requires remodeling of endothelial junctions.33,45JAM-C may also be necessary for the formation and maintenance of different cell junctions as it colocalizes at cell-cell contacts with adherence and tight junction proteins.19,33,46,47JAM-Cmediated cell polarization has been proposed as the underlying mechanism for its functions.39JAM-C directly associates with the cell polarity protein PAR-3, targeting it to tight junctions.31Furthermore, JAM-Cmutant mice are infertile due to a defect in spermatid differentiation, which requires polarization of round spermatids.22JAM-C appears to be essential for the assembly of a cell polarity complex containing PAR-6, aPKC, PATJ, and the small GTPase Cdc42 that ensures elongation and maturation of spermatids. Here, we investigate the role of JAM-C in hematopoiesis. We demonstrate that JAM-C is usually expressed on hematopoietic progenitors and that expression levels decrease with loss of self-renewal and increased differentiation. Deletion ofJam-Cin mice resulted in increased bone marrow (BM) cellularity caused by an increase in myeloid progenitors and granulocytes. Our phenotypic analysis combined with in vitro and in vivo characterization provides evidence that JAM-C is usually involved in the differentiation of HSCs into myeloid progenitors. == Methods == == Mice == Female C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). The congenic strain Igha B6 Ptprca B6.SJL used for transfer experiments was generated by crossing B6.SJL-Ptprca Pepcb/BoyJ with B6.Cg-Igha Thy1a Gpi1a/J (The Jackson Laboratory, Bar Harbor, ME).Jam-C/mice were generated a5IA by Lexicon Genetics (Woodlands, TX) by homologous recombination. Gestational age of heterozygous pregnancies was determined by detection of the vaginal plug as embryonic day (E) 0.5. All animals were housed in microisolators in a specific pathogenfree facility. Animal experiments were approved by the Institutional Animal Care and Use Committee of Genentech. == Reagents == JAM-Cspecific antibodies were generated by Josman (Napa, CA). a5IA In brief, rabbits were immunized with recombinant his-tagged murine JAM-C ectodomain. JAM-C reactive antibodies were obtained from the bleeds by affinity purification. == Cell isolation == BM cells were obtained from mouse femurs and tibia by flushing the central cavity with Dulbecco altered Eagle medium (DMEM) supplemented.