Neuregulin found in cultured-sciatic nerve conditioned medium causes neuronal differentiation of PC12 cells.
The present work deals with the search and identification of the molecule or combination of molecules, present in a medium conditioned by cultured rat-sciatic nerves (CM), able to cause neuronal differentiation of PC12 cells. The molecular mass range of the active fraction, as well as the thermostability and heparin affinity of the active component found in previous work, all characteristics shared with neuregulin (NRG) family members, led us to search for a NRG protein in the CM. Nerves were previously cultured for 8 days and the CM collected every 24 h, the following 3 days. The CM was concentrated (30,000 NMWL) and fractionated by quaternary ammonium chromatography and Cibacron blue affinity chromatography. The most active fraction B1.2 was further characterized by heparin affinity chromatography, size exclusion HPLC, Western blotting and immunoprecipitation. Results reveal abundance of NRG mRNA in the cultured nerves, presence of a 54 kDa NRG protein in the CM that increases along fractionation, and progressive diminution of fraction B1.2 differentiation activity on PC12 cells by gradual removal of the NRG protein by immunoprecipitation. The abundance of Schwann cells and the lack of axons in the cultured nerves suggest Schwann cells as the main NRG source, to which fibroblasts and perineurial cells might contribute.
Villegas
R
R
Centro de Biociencias, Instituto de Estudios Avanzados, Caracas, Venezuela. rvillega@reacciun.ve
Villegas
G M
GM
Longart
M
M
Hernández
M
M
Maqueira
B
B
Buonanno
A
A
García
R
R
Castillo
C
C
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002458:Cell Fractionation / Q000379:methods; D002846:Chromatography, Affinity; D004396:Coloring Agents; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D015870:Gene Expression / Q000502:physiology; D006493:Heparin; D020880:Neuregulins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D009474:Neurons / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D016716:PC12 Cells; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012583:Schwann Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D012584:Sciatic Nerve / Q000166:cytology*; D014227:Triazines
D004396:Coloring Agents; D017077:Culture Media, Conditioned; D020880:Neuregulins; D012333:RNA, Messenger; D014227:Triazines; C012842:Cibacron Blue F 3GA; D006493:Heparin