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 identif ied in most tissues, including the central nervous system, where the m ajor IGFBPs have been localized. The regulation and roles of IGFBPs in IGF action in the developing brain remain unclear. In this study we e xamined the expression and anatomical distribution of IGFBP messenger RNAs (mRNAs) in the newborn rat olfactory bulb (OB) during the first p ostnatal week. We used our recently developed newborn rat OB organ cul ture system, which emulates the first week of in vivo development, to identify and characterize expressed and secreted IGFBPs and to determi ne the role of the local growth factors IGF-I and basic fibroblast gro wth factor (bFGF) in their regulation. Postnatal day 1 rat OBs were cu ltured 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 sho wn by us to maintain morphology and differentiation of neuronal and gl ial cells. Conditioned medium was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western ligand blotting using [ I-125]IGF-I, and IGFBPs were characterized by immunoprecipitation. Wes tern ligand blotting of conditioned medium revealed two bands at 24 ki lodaltons (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-k Da bands only. In combination, IGF-I and bFGF enhanced all four bands above that seen with either growth factor alone. Total RNA was extract ed from fresh day 1, day 6, and cultured OBs for Northern blotting usi ng 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 mR NA showed lower expression than at day 1, and IGFBP-5 mRNA was similar ly expressed. When day 1 OBs were cultured for 6 days, mRNA was also r eadily detected for IGFBP-2, -4, and -5, but not for IGFBP-3. All dete cted mRNA species were enhanced by IGF-I. Basic FGF enhanced IGFBP-2 m RNA whether alone or in combination with IGF-I and enhanced only IGFBP -2 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 transcr iption of IGFBP and IGF-I mRNAs were located by in situ hybridization in both fresh and cultured bulbs. IGFBP-5 mRNA was located in fresh OB s at D1, with increased expression by day 6 in similar overlapping are as of the glomerular layer (GL; mainly populated by glial cells) and o n the overlying OB meninges. Similar expression was seen in IGF-I- and bFGF-treated cultured OBs. IGFBP-4 mRNA was expressed on overlying me ninges in both day 1 and day 6 OBs, with scattered expression in the G L, which was more evident in cultured OBs. IGFBP-5 mRNA was also expre ssed in both fresh and cultured OB in the neuron-rich mitral cell laye r and the outer GL. IGFBP-3 mRNA was detectable only in cultured OB, o n the periphery of the GL. IGF-I mRNA expression was seen in glomerula r and mitral cell layers in fresh and cultured OB, thus overlapping al l sites of IGFBP mRNA expression. We conclude that IGFBP-2, -4, and -5 are locally expressed in the developing rat OB, both in vivo and in v itro at least during the first 6 postnatal days. Their distinct anatom ical locations, which overlap with IGF-I mRNA expression sites, sugges t a role for IGFBP in modulating the paracrine actions of IGF-I. Expre ssion of IGFBP-3 mRNA and protein, only seen in cultured bulb, may rep resent an in vitro injury response. IGFBP mRNA expression and their pr otein levels are differentially regulated by IGF-I and bFGF, alone or in combination. These findings support a role for locally synthesized IGFBPs in discrete regions of developing brain. The modulation of thei r expression provides further mechanisms of interaction between the lo cally synthesized growth factors IGF-I and bFGF.