title: Role of stromal cells and macrophages in fibronectin biosynthesis and matrix assembly in human long-term marrow cultures.
abstract: Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.
authors.affiliation: INSERM/CRTS, Besançon, France.
authors.forename: H
authors.initials: H
authors.lastname: Lerat
authors.forename: J C
authors.initials: JC
authors.lastname: Lissitzky
authors.forename: J W
authors.initials: JW
authors.lastname: Singer
authors.forename: A
authors.initials: A
authors.lastname: Keating
authors.forename: P
authors.initials: P
authors.lastname: Herve
authors.forename: P
authors.initials: P
authors.lastname: Charbord
mesh_terms: D001853:Bone Marrow / Q000502:physiology*; D001854:Bone Marrow Cells; D002478:Cells, Cultured / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000737:chemistry; D005353:Fibronectins / Q000096:biosynthesis* / Q000235:genetics; D005455:Fluorescent Antibody Technique; D014644:Genetic Variation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D008264:Macrophages / Q000502:physiology*; D011233:Precipitin Tests
chemical_list: D003470:Culture Media; D005353:Fibronectins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor