Laminin B1 expression is required for laminin deposition into the extracellular matrix of PC12 cells.
The extracellular matrix of rat pheochromocytoma PC12 cells was shown by indirect immunofluorescence to consist of a network of fibronectin. The matrix did not contain laminin. The cells synthesized messenger RNA for fibronectin, laminin B2, and s-laminin but not for entactin or the B1 and A chains of laminin. Laminin B2 but not laminin B1 was detectable in the culture medium and in cell lysates. A full-length cDNA clone for the B1 chain of laminin was constructed in the plasmid p-444, which contains the neomycin-resistance marker and human beta-actin promoter. PC12 cells were transfected with this recombinant plasmid, and stable neomycin-resistant clones were isolated and characterized. Clones that synthesized laminin B1 messenger RNA were found to deposit a laminin-containing matrix. In many of these clones the deposition of the fibronectin matrix was greatly diminished. The laminin matrix was predominantly localized in the intercellular spaces forming a honeycomb pattern. The morphology of the laminin-synthesizing transfected cells was markedly different from the parental cells. The cells grew in tight clusters that were resistant to dissociating agents. It is concluded that the B1 chain of laminin contains information that is required for the formation of a stable laminin-containing extracellular matrix network either by interaction with cell surface receptors or other extracellular matrix components. Furthermore, expression of the laminin B1 gene may be a central regulatory point in determining extracellular matrix composition during embryogenesis.
Reing
J
J
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
Durkin
M E
ME
Chung
A E
AE
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002999:Clone Cells; D015342:DNA Probes; D005109:Extracellular Matrix / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D007797:Laminin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D016716:PC12 Cells; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger; D014162:Transfection
D015342:DNA Probes; D007797:Laminin; D046911:Macromolecular Substances; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA