Identification, localization, and regulation of insulin-like growth factor binding proteins and their messenger ribonucleic acids in the newborn rat olfactory bulb.
Insulin-like growth factor binding proteins (IGFBPs) have been identified in most tissues, including the central nervous system, where the major IGFBPs have been localized. The regulation and roles of IGFBPs in IGF action in the developing brain remain unclear. In this study we examined the expression and anatomical distribution of IGFBP messenger RNAs (mRNAs) in the newborn rat olfactory bulb (OB) during the first postnatal week. We used our recently developed newborn rat OB organ culture system, which emulates the first week of in vivo development, to identify and characterize expressed and secreted IGFBPs and to determine the role of the local growth factors IGF-I and basic fibroblast growth factor (bFGF) in their regulation. Postnatal day 1 rat OBs were cultured serum free for 6 days in the absence or presence of IGF-I (150 ng/ml) and bFGF (25 ng/ml), alone or in combination, as previously shown by us to maintain morphology and differentiation of neuronal and glial cells. Conditioned medium was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western ligand blotting using [125I]IGF-I, and IGFBPs were characterized by immunoprecipitation. Western ligand blotting of conditioned medium revealed two bands at 24 kilodaltons (kDa) and 30 kDa and a doublet at 38-42 kDa. All bands were enhanced by IGF-I treatment, whereas bFGF enhanced the 24-kDa and 30-kDa bands only. In combination, IGF-I and bFGF enhanced all four bands above that seen with either growth factor alone. Total RNA was extracted from fresh day 1, day 6, and cultured OBs for Northern blotting using complementary DNA probes for IGFBP-2, -3, -4, and -5. In fresh day 1 OBs, mRNA was detected for IGFBP-2, -4, and -5, but not for IGFBP-3. In fresh day 6 OBs IGFBP-2 mRNA was more abundant, whereas IGFBP-4 mRNA showed lower expression than at day 1, and IGFBP-5 mRNA was similarly expressed. When day 1 OBs were cultured for 6 days, mRNA was also readily detected for IGFBP-2, -4, and -5, but not for IGFBP-3. All detected mRNA species were enhanced by IGF-I. Basic FGF enhanced IGFBP-2 mRNA whether alone or in combination with IGF-I and enhanced only IGFBP-4 mRNA when given alone. IGFBP-5 mRNA was not affected by bFGF alone, but its enhancement by IGF-I was attenuated by bFGF. Sites of transcription of IGFBP and IGF-I mRNAs were located by in situ hybridization in both fresh and cultured bulbs.(ABSTRACT TRUNCATED AT 400 WORDS)
Russo
V C
VC
Centre for Hormone Research, Royal Children's Hospital, Parkville, Victoria, Australia.
Edmondson
S R
SR
Mercuri
F A
FA
Buchanan
C R
CR
Werther
G A
GA
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D003720:Densitometry; D016222:Fibroblast Growth Factor 2 / Q000502:physiology; D005786:Gene Expression Regulation; D017403:In Situ Hybridization; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000502:physiology; D009830:Olfactory Bulb / Q000737:chemistry* / Q000378:metabolism; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2; D007334:Insulin-Like Growth Factor I