Regional distribution and developmental expression of epidermal growth factor and transforming growth factor-alpha mRNA in mouse brain by a quantitative nuclease protection assay

Abstract

A solution-hybridization ribonuclease-protection assay was used to identify epidermal growth factor (EGF) mRNA in mouse brain and to compare the regional and developmental levels of EGF gene expression in the CNS with those of its structural homolog, transforming growth factor-alpha (TGF-alpha). Adult brain regions examined included brainstem, cerebellum, cerebral cortex, hippocampus, basal hypothalamus, olfactory bulb, olfactory tubercle, striatum, and thalamus. While both EGF and TGF-alpha mRNAs were detected in all regions, TGF-alpha mRNA levels were 15-170 times higher, ranging from 0.39 (cerebellum and cerebral cortex) to 2.93 (striatum) pg TGF-alpha mRNA/micrograms total cytoplasmic RNA. In contrast, EGF mRNA levels ranged from 11 to 36 fg EGF mRNA/micrograms, with the highest regional concentrations observed in olfactory bulb, basal hypothalamus, and cerebellum. In our comparison between sexes, no significant male-female differences in EGF or TGF-alpha mRNA levels were observed for any region of adult brain. However, in the pituitary gland, consisting of both endocrine and neural elements, EGF and TGF-alpha mRNA levels were significantly higher in males (234 and 215 fg/micrograms, respectively) than in females (172 and 118 fg/micrograms, respectively). An examination of growth factor gene expression in the developing CNS revealed EGF and TGF-alpha mRNAs detectable as early as embryonic day 14 (earliest time point studied). While gene expression for both peptides continued into the postnatal period, EGF and TGF-alpha mRNA levels were nearly equal to adult concentrations by postnatal day 10. Taken together, our findings provide evidence for the synthesis of EGF in brain and suggest a role for both EGF and TGF-alpha in the development and support of the mammalian CNS.

Reduced brain-derived neurotrophic factor in prefrontal cortex of patients with schizophrenia

“…28 Ribonuclease protection assay The RNA samples from the control group and patients with schizophrenia were processed randomly and the experimenter was blind to diagnostic group during the assay and data acquisition. The ribonuclease protection assay (RPA) was performed as detailed previously 52 except with a reduced concentration of RNase A (10 mg/ml). Total RNA (20 mg) was hybridized with the antisense probe for BDNF, whereas 2 mg of total RNA was used for the cyclophilin RPA.…”

Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the α6-integrin subunit

“…The method of Blum (Blum, 1989;Lazar and Blum, 1992;Weickert and Blum, 1995) was used to quantitate levels of mRNA transcripts specific for preproEGF, preproTGF-a, or EGFR. Briefly, 50 pg of 32 P-labeled antisense riboprobe (about 50,000 cpm) were added to known amounts of sense transcripts (range: 0-5.0 pg) to prepare the standard series, or to the 30 µg aliquots of SMG sample total RNA in 30 µl of hybridization buffer (see above).…”

“…For quantitation of preproEGF mRNA we used pmEGF 344 (Lazar and Blum, 1992), containing a 344 bp cDNA insert corresponding to bases 3,023-3,366, coding for the mature EGF molecule (Gray et al, 1983). Restriction with EcoRI and transcription by T7 RNApolymerase yields a 360 nt sense fragment; restriction with SpeI and transcription by T3 RNA-polymerase produces a 407 nt antisense fragment.…”

Transforming growth factor-alpha stimulates proliferation of mammotrophs and corticotrophs in the mouse pituitary

“…In the case of the growth of pituitary cells, transforming growth factor-(TGF-) is one of the candidates for the oestrogen-induced growth factors (Borgundvaag et al 1992). TGF-is produced in pituitary glands of several species (Kudlow & Kobrin 1984, Kobrin et al 1987, Lazar & Blum 1992. In rat pituitary cells, TGF-mRNA expression was detected in somatotrophs, gonadotrophs and mammotrophs .…”