316

Molecular Brain Research, 16 (1992) 316-322 ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00

BRESM 70526

Comparison between epidermal growth factor, transforming growth factor-c and EGF receptor levels in regions of adult rat brain Matthew R. Kaser, Jayaraman Lakshmanan and Delbert A. Fisher Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, UA 90509 (USA) (Accepted 28 July 19921

Key words: Brain; Growth factor; Receptor; lmmunoreactivity; Reverse transcriptase-PCR

Examination of adult rat brain regions by specific radioimmunoassays revealed a widespread distribution of transforming growth factor-c~ (TGF-a), but not epidermal growth factor (EGF), the peptide that had previously been reported to be present in rodent brain. Polyadenylated R N A samples from the different regions of rat brain were analyzed by Northern blot to identify m R N A species encoding precursor proteins for EGF (preproEGF), TGF-o~ (preproTGF-a), and the E G F / T G F - ~ receptor. The results indicate that T G F - a is the most abundant ligand for the E G F / T G F - a receptor in most parts of the brain analyzed. Message for preproEGF was only detectable after prolonged autoradiographic exposure; levels of preproEGF m R N A were between two and three orders of magnitude lower in brain than those expressed in control tissue (kidney), and one to two orders of magnitude lower than preproTGF-a m R N A levels in all brain regions. These results were confirmed by analysis of m R N A by R T / P C R , and support the hypothesis that expression of preproEGF m R N A in the brain is limited to smaller discrete areas, whereas preproTGF-a gene expression is almost ubiquitous.

INTRODUCTION

Epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) are members of the epidermal growth factor family of proteins, and are potent mitogens for a variety of epithelial cell types ~'. TGF-o~ exhibits 35% amino acid homology to EGF and competes with similar affinity as EGF for binding to EGF receptor (EGF-R); EGF and TGF-a peptides are derived from the precursor proteins preproEGF and preproTGF-a. Evidence for the existence of EGF-R immunoreactivity 22, TGF-c~ immunoreactivity t~,20, and preproTGF-c~ m R N A ~,,32 in mammalian brain have been reported previously. However, evidence for the presence of EGF immunoreactivity in the brain remains controversial 10,18,27,3fl In the present study, we measured the distribution of EGF and TGF-a immunoreactivities in brain cortex, cerebellum, midbrain, and brain stem by specific radioimmunoassays (RIA) developed in our laboratory. To corroborate the RIA data we examined the distribution of m R N A species encoding preproTGF-a, preproEGF, and EGF-R in the adult rat brain regions.

The results reveal that TGF-e~ is more widespread in brain than EGF. The localization of EGF-R m R N A in brain supports the evidence that TGF-o~ or its precursor may utilize the EGF-R signalling pathway. MATERIALS

AND METHODS

Animals Sprague-Dawley adult male rats (150 3110 g) were purchased from Harlan Laboratories, and were housed according to established guidelines. Food and water were available ad libitum. Extraction of brain and t'arious region.~ ]br peptide measurement Animals were killed by decapitation. Whole brain and brain regions including cerebral cortex, cerebellum, midbrain, and brain stem were separated, weighed, and frozen on dry ice. They were subsequently homogenized (1:2, w / v ) in phosphate buffered saline (PBS, 511 mM phosphate, pH 7.4) using a tissuemizer. The homogenates were centrifuged at 100,000× L' for 60 rain. The supernatants were stored at - 7 0 ° C until assay. Generation and characterization o] E(;F attd TGF-alpha antisera EGF was isolated from adult male rat submandibular glands (SMG-EGF) 2'~. Both rat E G F and antiserum were kindly provided by Dr. W.D. Odell of the University of Utah School of Medicine. T G F - a antiserum was generated in rabbits in our laboratory using chemically synthesized TGF-~ as immunogen 4. The specificities of the antisera were characterized by immunoblotting.

Correspondence: D.A. Fisher, Department of Pediatrics, Harbor-UCLA Medical Center, lIl(J0 West Carson Street, Torrance, ( ' A 90509, USA.

317 For these studies, 20 /xg of EGF and 20 /xg TGF-a were first separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, 15% acrylamide) under reducing conditions according to Laemmli 17. The peptides were electrophoretically transferred from the gel to an Immobilon-P (Millipore) membrane in 25 mM Tris, pH 8.3, 192 mM glycine, 20% methanol, 0.1% SDS, as described by Towbin et al. 31. Individual strips containing the peptides were immunoblotted as described previously 19. The immunoreactive bands were detected by autoradiography after labelling with 12SI-goat anti-rabbit IgG.

EGF and TGF-a radioimmunoassays EGF and TGF-a were measured by liquid phase, double antibody RIA systems 4. Purified rat submandibular gland (SMG) EGF and synthetic TGF-a were utilized for iodination and reference standard in the respective assays 4. For the EGF-RIA, the antiserum was used at a final dilution of 1:300,000; the final dilution of antiserum for the TGF-a was 1:15,000. Assay sensitivity is defined as the concentration of unlabeled ligand required to cause a significant ( > 10%) displacement of labeled growth factor peptide. For tests of parallelism, pooled brain extracts were used. Recovery studies were performed by adding known amounts of growth factor to three different concentrations of brain extracts. Pooled brain extracts were utilized for determinations of both withinand across-assay coefficients of variation. RIA results were calculated using log-logit transformation.

RNA isolation Brains from between four to six rats were dissected following decapitation, and the cortex, cerebellum, midbrain, and brain stem were separated. Tissues were snap frozen in liquid nitrogen and stored at - 70°C for up to one month. Total RNA was isolated from tissue (n = 4-6) by the method of Chomszynski and Sacchi 7 and quality assessed by comparisons of O.D. absorbance at 260 and 280 nm. Between 3 and 5 mg (determined by absorbance at 260 nm) total RNA was enriched for polyadenylated RNA (poly(A) + RNA) using oligo d(T)-cellulose affinity chromatography 1.

Northern blot hybridization Approximately 15 ~g poly(A) + RNA was loaded and separated on 1.2% agarose gels containing 6.5% formaldehyde; RNA was transferred to nylon filters with 20 × SSC (1 × SSC is 0.15 M NaC1, 15 mM sodium citrate), and the filter prehybridized at least 1 h in 50% formamide at 42°C. In order to prevent increased autoradiographic background following successive hybridizations of one filter, we separated identical quantities of pooled sample on three agarose gels and transferred RNA to nylon filters (Magna, MSI, Westboro, MA 01581) for each DNA probe used for analysis. Following exposure, we stripped one filter (EGF-R filter) by boiling twice in 0.1 ×SSC, 0.1% sodium dodecyl sulphate (SDS), and re-hybridized with a rat beta-actin cDNA probe as control. Filters containing 5/xg or 2.5 ~zg rat kidney poly(A) + RNA were probed in parallel with those of rat brain (15 p~g) and rat liver (15/xg) poly(A) + RNA samples. Northern analysis of three separate preparations was performed; data are presented from one typical experiment. All probes were prepared as follows (rat preproEGF, gift from Dr D. Dorow, Melbourne s; rat preproTGF-a, gift from Dr D. Lee, University of North Carolina, Chapel Hill 21; rat EGF-R, gift from Dr S. Earp, Univ. North Carolina 25; rat fl-actin 24). Fifty ng of DNA was labeled with [a-3:p]dCTP to a specific activity of at least 109 cpm per ~g DNA (Multi-prime labeling kit, Amersham International, Arlington Heights, IL 60005). The probe was precipitated with ethanol, resuspended in water, and boiled for two minutes. The melted probe was added to the pre-hybridization medium and hybridized to each of the three filters for at least 20 h at either 42°C (preproEGF, or EGF-R), or 37°C (preproTGF-a). The filters were washed (2×SSC, 0.1% SDS, at 50°C) and exposed to X-OMAT film ( - 70°C with intensifying screens) as described in the legends for the figures. The autoradiographs were analyzed densitomerically and data normalized to/3-actin mRNA densities.

Linear sensitivity of the X-OMAT film was determined by analysis of preproEGF mRNA bands from two different lanes (loaded with 2.5 tzg and 5.0 /~g, estimated by absorption at 260 nm) of rat kidney poly(A) + RNA separated on the same gel, transfered to nylon membrane, and hybridized with the preproEGF probe as described above. After 1 h exposure, the optical density (OD) values of the two bands were determined using a BioRad 620 Video Densitometer with the following results. 2.5 p.g mRNA = 0.14 OD units; 5.0 /xg mRNA = 0.26 OD units. The area under the curve (OD.mm) was determined to be proportional to the OD (0.447 OD.mm units, and 0.937 OD.mm units, respectively). All other OD measurements fell between 0.12 and 0.86 OD units, with the exception of the rat liver EGF-R band (1.22 OD units).

PCR One /zg of poly(A) + RNA was transcribed into cDNA using AMV reverse transcriptase and poly d(T) as primer; cDNA was isolated by phenol, phenol-chloroform-isoamyl alcohol extraction, followed by isopropanol precipitation. The cDNA was taken up into 50 txl water, and 5 /zl used for PCR amplification. PCR primers (including endonuclease restriction sites) were synthesized according to the published sequence for rat preproTGF-a 3,13 or for human and mouse preproEGF cDNA 2'21, and were designed such that there would be no primer cross-hybridization to either growth factor cDNA species. PreproTGF-a primers were: sense, 5'-TGC CCA GAT TCC CAC ACT CAG-3' (rat preproTGF-a residues 47-53); antisense, 5'-CTT CAG GCG GGC GCT GGG CTF CTC GTG-3' (rat preproTGF-a residues 148-140). PreproEGF primers were: sense, 5'-CAA GGC AGC ATG CTG AAG CCC TCG-31 (mouse preproEGF residues 728-735); antisense, 5'-GAC CAC AAA CCA A G G T r G GGG ACA AGA-3' (mouse preproEGF residues 11071099). PCR cycles were as follows: cDNA template was melted for 1 min at 94°C, annealed for 1 min at 57°C (preproTGF-a) or 51°C (preproEGF), and extension for 2 min at 72°C. The reaction was allowed to proceed during the first cycle without Taq DNA polymerase until the annealing temperature had been reached in order to maximize template-primer specificity; a total of thirty-five cycles were completed. The DNA fragments were separated on 5% acrylamide gels and stained with ethidium bromide. Control plasmid containing the human preproTGF-a cDNA was analyzed in parallel and resulted in a DNA fragment of the expected size.

RESULTS

Growth f a c t o r peptides in brain Rabbit peptides

antisera were

raised

tested

for

against their

munoreactivity with both EGF 1:1000

dilutions using Western

bilon-P membrane

EGF

and

specificity

and TGF-a

TGF-a and

im-

antigens at

blot analysis. Immo-

bearing purified SMG-EGF

showed

a single band with an approximate molecular mass of 6 kiloDaltons detected

(6 k D a )

when

(Fig.

1, l a n e A ) . N o b a n d

a similar membrane

EGF was incubated with TGF-a B). T h e m e m b r a n e intense radiolabeled

was

containing SMG-

a n t i s e r u m ( F i g . 1, l a n e

bearing TGF-a

also produced

an

band with the expected apparent

m o l e c u l a r m a s s o f 5.6 k D a u p o n i n c u b a t i o n w i t h T G F - a a n t i s e r u m (Fig. 1, l a n e C). N o b a n d w a s o b s e r v e d w h e n an identical membrane serum

(Fig.

1, l a n e

was incubated with EGF D).

Both

antisera

showed

antihigh

specificity towards respective antigens. Representative EGF and TGF-a

s t a n d a r d c u r v e s a r e s h o w n in Fig. 2 A

a n d 2B, r e s p e c t i v e l y . T h e m e a n

(± S.E.M.) sensitivity

318 EGF

i

I

l

0

r~

I

TGF- olpho

l

l

l

250 E o

200

~

150

44.2

I-- 2 9 . 2 -1L9 hi 18.1

o 100 Q. o EL SO F-

14.4

E