Comparison between epidermal growth factor, transforming growth factor-α and EGF receptor levels in regions of adult rat brain

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

<[ J

0

D 0 Ld J 0

CORTEX

5.6

CEREBELLUM

MIDBRAIN

BRAINSTEM

Fig. 3. TGF-~ immunoreactivity in brain regions. The different brain regions were homogenized in PBS, pH 7.4, centrifuged and the supernatants were used for quantitation of TGF-alpha and protein concentrations as described in the text. TGF-c~ concentration (mean: hatched bar: S.E.M.: filled bar) is expressed as pg per mg protein.

2.9

Strip:

A

B

C

T h e d i s t r i b u t i o n o f TGF-t~ a n d E G F

D

Fig. 1. Characteristics of anti-rat EGF and anti-rat TGF-oe polyclonal antisera by Western blot analysis. 20 ttg of purified rat SMG-EGF (lanes A and B) or synthetic TGF-oL (lanes C and D) were transferred by electrophoresis to Immobilon-P membrane and reacted with anti-rat EGF antiserum (A, D), or with anti-rat TGF-tt antiserum (B, C). Both antisera were used at 1:1000 dilutions as described in the Materials and Methods. Lanes A and B: autoradiographs of Immobilon-P membrane bearing SMG-EGF reacted with anti-rat EGF and anti-rat TGF-~ antisera respectively. Lanes C and D: autoradiograph of lmmobilon-P membrane bearing synthetic TGF-a reacted with anti-rat TGF-a and anti-rat EGF antisera respectively. Lanes A and C were exposed for 2 days, whilst lanes B and D were exposed for 6 days to X-ray (Kodak X-OMAT) film.

immunoreac-

tivity in b r a i n r e g i o n s was e x a m i n e d by R I A ; t h e results a r e s h o w n in Fig. 3. All b r a i n r e g i o n s c o n t a i n e d TGF-c~, t h e c o n c e n t r a t i o n o f w h i c h fell w i t h i n t h e r a n g e o f t h e standard

curve; E G F

was not p r e s e n t

at d e t e c t a b l e

levels ( R I A d a t a not shown).

Messenger RNA levels in the brain Comparative mRNA

a n a l y s e s f r o m a d u l t rat cortex,

c e r e b e l l u m , m i d b r a i n , a n d b r a i n s t e m a r e s h o w n in Fig. 4. T h e a u t o r a d i o g r a p h i c b a n d s w e r e a n a l y z e d by d e n s i t o m e t r y , a n d t h e d a t a w e r e n o r m a l i z e d to b e t a - a c t i n b a n d d e n s i t i e s s h o w n in "Fables [ a n d ll. V e r y little

of the EGF and TGF-a pg/tube,

assays w e r e 25 _+ 7 a n d 8 _+ 2

r e s p e c t i v e l y . S e r i a l d i l u t i o n s o f a d u l t rat b r a i n

p r o d u c e d a p a r a l l e l d i s p l a c e m e n t line to TGF-c~, b u t not to E G F

standard

in t h e R I A . In c o n t r a s t to rat

b r a i n e x t r a c t , rat u r i n e p r o d u c e d

a parallel displace-

preproEGF

(Fig. 4, l a n e Extended

w e r e p r e s e n t in m i d b r a i n

in b r a i n s t e m

autoradiographic

(Fig. 4, l a n e 5).

e x p o s u r e o f this filter remRNA

which con-

99 •

95 H

)el/tube 20 I : t

-" 40 I

o

-" &

-"

60 80 t00 I I 1 0 )

-"

Adult Rot Brein E x t r a c t (1:2 w/v)

95

200 I Adult Rot Urine

NI ADULT RAT B R A I N E X T R A C T 20 40 60 80 I00 200

9C

80

d

4) a n d

vealed the presence of preproEGF

99

70 60 50 40 30 20

was d e t e c t e d in c o r t e x o r c e r e b e l -

f i r m e d this r e s u l t ( n o t shown). P r e p r o T G F - c ~ e x p r e s -

m e n t line in t h e E G F - R I A .

90

mRNA

lum; h i g h e r levels o f m R N A

8C

~ 5o

) EGF Standard

10

4o

TGF- oiphe Slondord

~ "t..

3O 2O tO

I000

I0 E G F (pgllube)

TGF

olpho Ipg/tube)

Fig. 2. A: r e p r e s e n t a t i v e d i s p l a c e m e n t line g e n e r a t e d by i n c r e a s i n g a m o u n t s of S M G - E G F , rat brain extract, and ral urine in the E G F - R I A . B:

representative displacement line generated by increasing amounts of synthetic TGF-(~ and rat brain extract in the TGF-c~ RIA. Each point represents the mean + S.E.M. (S.E.M. fall within area covered by point) of three individual determinations.

319 TABLE I [3-Actin mRNA leceb in tissue

Filters supporting 5 /,tg (kidney) or 15 /~g poly(A) + R N A were probed with a 1200 bp Bgll fragment of rat /3-actin; filters were exposed to X - O M A T film for 2 days, and bands analyzed by densitometry. The area under the curve ( O D . m m ) was judged to be proportional to m R N A levels. mRNA lecels per 15 # g poly(A) + RNA (OD. m m units) Kidney Lit 'er

Cortex

Cerebellum

Midbrain

Brah~stem

10.23

1.32

0.35

4.23

2.80

0.44

sion was detected in cerebellum, brainstem, midbrain, and cortex. Messenger RNA levels of the 9.5 kilobase (9.5 kb) EGF/TGF-c~ receptor species was similar to that of p r e p r o T G F - a in each region of brain studied (Table II). Analysis using the E G F / T G F - a receptor cDNA probe confirmed previous reports that several m R N A species encoding the receptor are present in rat t i s s u e 14"23'25. In contrast to the results from kidney and liver, a 9.5 kb species is the major mRNA component in the brain, particularly of the midbrain and brainstem; 5.0 kb and 6.8 kb m R N A species are also present in lower quantities in all brain regions. PCR analysis of cDNA transcribed from brain poly(A) + RNA using rat T G F - a primers indicated that DNA fragments of expected size (320 bp), equivalent in size to those synthesized from a plasmid containing genuine human p r e p r o T G F - a cDNA were present at 1

2

3

4

5

6 kb

28ST G F - o~

I

"~ 18S-

28s-

.....

~ i~:,,

-4.5

I L ~

|

! -5.0

EGF 18S-

similar levels in all regions of the brain (data not shown). No DNA fragments were detected in brain PCR samples incubated with p r e p r o E G F primers; this might be due to species differences in rodents, or to annealing affinities as discussed below. The primers were synthesized on the basis of regions of DNA identity between human and murine p r e p r o E G F cDNA sequences, since the full length rat p r e p r o E G F cDNA sequence is not available. There are two possible reasons for reduced t e m p l a t e / p r i m e r affinity: (i) small differences in the DNA sequence may alter primer affinity for template, or (ii) the region chosen for amplification might adopt unknown tertiary structures, thus impeding PCR. In separate experiments using mouse E G F primers, a faint band of the expected size (1100 bp) was seen after ethidium bromide staining of PAGE-analyzed PCR reactions containing rat kidney or liver cDNA as control. This suggested that one or both primers might not anneal efficiently to the template; the result was replicated when we performed PCR over a range of annealing conditions (45-65°C; data not shown). Sequencing of the TGF-ot PCR fragments was found not to be possible due to technical difficulties associated with PCR-generated DNA fragments, but restriction enzyme digestion of the R T / P C R DNA produced fragments of the expected size as deduced from the published cDNA. Southern blot analysis using rat

--9.5

-6.8

28SEGF-R

m 18S-

-"5.0 -3.0 -2.4

Fig. 4. Filters probed with rat TGF-o~ c D N A fragment (1.3 kb 3' non-coding region of rat preproTGF-a), rat p r e p r o E G F c D N A fragment (1.7 kb B a m - H l - P s t I fragment corresponding to p r e p r o E G F nucleotides 1304-3062), rat E G F - R c D N A fragment (2.3 kb E c o R 1 - E c o R 1 5' region including signal peptide to t r a n s m e m b r a n e domains); lane 1: kidney; 2: brain cortex; 3: cerebellum; 4: midbrain; 5: brainstem; 6: liver. 15 p.g R N A were loaded in each lane except kidney (5 # g , TGF-c~ and EGF-R; 2.5 /.tg EGF). kb refers to the m R N A species transcript size in kilobases. Positions of rat liver ribosomal R N A size markers (28S and 18S) electrophoresed in an adjoining lane, visualized with ethidium bromide staining, are indicated. Exposure times: TGF-o~, 2 days (lanes 1-6); EGF, 2.5 h (lane 1), or 1 day (lanes 2-6); EGF-R, 6 days (lane 1), or 1 day (lanes 2-6).

T A B L E II Growth factor and receptor mRNA lecels in tissue relatice to [3-actin lel "els

N u m b e r s expressed as the ratio of growth factor or receptor m R N A O D - m m to /3-actin m R N A O D - m m . /3-Actin = 1.000 arbitrary unit. Tissue

Kidney Liver Cortex Cerebellum Midbrain Brainstem

mRNA species PreproTGF- a 4.5 kb

PreproEGF 5.0 kb

EGF-R 9.5 kb

0.208 2.598 0.937 2.758 1.192 2.007

1.779 0.033 0.005 0.1)14 0.003 0.005

0.301 15.650 I.I 19 1.400 0.769 1.223

320 T G F - a c D N A probe indicated positive hybridization to PCR fragments derived from rat brain, liver and kidney cDNA under stringent conditions (50% formaldehyde, 42°C: data not shown). DISCUSSION The Western blot analyses in the present study indicate that both the E G F and TGF-o~ antisera are highly specific for their respective antigens. The lack of cross-reactivity between TGF-o~ antiserum with E G F and vice versa enabled us to use the RIA systems to examine the concentrations of both factors in brain extracts. Although similar amounts of E G F and T G F - a were used for the immunoblot analyses, the TGF<~ band was more intense than the E G F band. This could be due to differences in the binding properties of T G F - a vs. E G F to the lmmobilon-P membrane, since Fig. 1 indicates that the antibody is sufficiently specific to react with detectable amounts of lmmobilon-Pbound EGF, and that T G F - a antibody does not appreciably cross-react, as demonstrated by extended autoradiography, with E G F peptide. Brain extracts did not displace the binding of radiolabeled E G F to antibody, suggesting that mature E G F either is absent, or that levels are below the detection limit of the RIA. Urine, however, produced thc expected parallel displacement in the E G F - R I A , suggesting that the antiserum is able to recognize the mature 6 kDa E G F peptide. The lack of EGF-immunoreactivity in rat brain is in contrast to our previous observations examining mouse brain extract ~s. In that earlier study, wc utilized a sensitive R I A that is specific for mouse EGF. These differences may thus be due to species specific processing of E G F precursor (preproEGF). It is also possible that the EGF-immunorcactivity identified by those experiments in mouse brain extract was mouse T G F - a . It should bc noted that E G F and TGF-c~ are highly homologous, and thus cross-reactivity is possible. Since mouse TGF-~ had not been isolated, we wcre not able then to confirm the specificity of the mouse E G F antiserum used in that study, however in the current study, we have demonstrated the specificity and lack of cross-reactivity of the antisera. PCR analysis showed that p r e p r o E G F was present in brain at levels below limits of detection when we used primers manifesting sequence identity between human and murine cDNAs. This might bc due to species differences within rodents, or to the possibility that the primers contained sequences that created conformational structures that might limit annealing at optimal temperatures. The TGF-c~ and E G F antisera used in this study are

highly specific since they show no cross-reaction when pure peptides were subjected to immunoblotting. Although the antisera could detect microgram quantities of E G F peptide by Western blot, our E G F - R I A did not detect immunorcactive material in rat brain extract, and thus we did not procede further to examine the brain tissue extracts by immunoblot analysis. We arc presently examining rat brain extracts with T G F - a antiserum to identify the nature of the immunoreactive species, In addition, we are investigating T G F - a localization by immunohistochemical techniques, and are in collaboration with others to identify sites of synthesis of p r e p r o T G F - a and p r e p r o E G F m R N A in subcortical regions of adult rat brain. The present data indicate abundant levels of E G F / T G F - c , receptor and p r e p r o T G F - ~ m R N A relative to p r c p r o E G F m R N A in adult rat brain cortex, cerebellum, midbrain, and brainstem. We were unable by PCR amplification to convincingly demonstrate significant levels of the p r e p r o E G F transcript in rat brain, although others have reported the presence of prep r o E G F message in mouse brain es. By Northern analysis, we observed that the p r e p r o E G F gene is transcribed primarily in the midbrain (Fig. 4, lane 4), to a lesser extent in brainstem (Fig. 4, lane 5), and at very low levels in other rat brain regions studied. Prolonged autoradiographic exposure of the filter (not shown) showed the presence of p r e p r o E G F m R N A in very low amounts, but this added little to the data. The data presented suggests that p r e p r o E G F messenger RNA is present in regional, localized areas, and that the m R N A levels may be diluted by non-preproEGF-transcribing tissue, and corroborates the protein data decribed for E G F localization in rat brain m. In agreement with this view, preliminary studies by Callaway et al. 5, described immunocytochemical localization of E G F to the globus pallidus of monkey brain; these authors also reported that T G F - a immunoreactivity was present in relatively larger amounts in nearly all other brain regions studied. A similar distribution of E G F and TGF-o~ is found in these regions of rat brain (Dr. S.E. Loughlin, personal communication). We were not able to determine the subcortical distribution of the messenger RNAs due to lack of suitable micro-dissecting equipment, but we are currently investigating this approach. Differential expression of preproTGF-c~ and the EGF/TGF-c~ receptor within different brain regions might reflect differences in neurological function; for example, cerebellu~m has more abundant preproTGF-~r and EGF/TGF-c~ receptor m R N A levels than other brain regions. Brain cortex m R N A levels of prep r o T G F - a and E G F / T G F - c t rcceptor were similar to those in midbrain tissue, whereas brain stem had pre-

321 p r o T G F - a m R N A levels i n t e r m e d i a t e to those in cereb e l l u m a n d cortex. C e r e b e l l a r p r e p r o E G F m R N A levels were only half the values expressed in liver, a n d a n o r d e r of m a g n i t u d e lower t h a n in kidney tissue. E G F / T G F - a r e c e p t o r m R N A was r e p r e s e n t e d by a p r e d o m i n a n t 9.5 kb b a n d in b r a i n tissue, in contrast to m u l t i p l e R N A species observed in o t h e r tissues.

CONCLUSIONS

E G F - R a n d preproTGF-o~ m R N A a b u n d a n c e s are similar in adult rat brain. P r e p r o E G F m R N A is two to three orders of m a g n i t u d e lower in a b u n d a n c e . T h e s e data are reflected in the levels of growth factor peptides analyzed by R I A . C e r e b e l l u m , in contrast with o t h e r regions, shows the greatest levels of R N A transcripts for both E G F - R a n d preproTGF-c~. T h e ratio of preproTGF-ce to p r e p r o E G F m R N A in b r a i n is the reverse of that d e t e c t e d in o t h e r tissues, such as submaxillary gland a n d kidney. A s s u m i n g a p r e d o m i n a n t a u t o c r i n e or p a r a c r i n e m e c h a n i s m of action, these data suggest that these growth factors have differential roles in various tissues. In addition, the processing a n d relative a m o u n t s of the E G F / T G F - c ~ receptor reflected by the different m R N A species in b r a i n c o m p a r e d to liver and kidney, suggests u n i q u e p o s t - t r a n s l a t i o n a l a n d p o s t - t r a n s c r i p t i o n a l processing of the r e c e p t o r in rat b r a i n tissue. T h e results p r e s e n t e d in this study suggest that T G F - a is the major ligand for the E G F - R in most regions of the adult rat b r a i n a n d that expression of p r e p r o E G F is m u c h more limited. Studies are in progress to identify the cellular localization a n d molecular n a t u r e of the TGF-o~ i m m u n o r e a c t i v i t y in brain. T h e f u n c t i o n s of E G F in the c e n t r a l n e r v o u s system are b e i n g investigated in various laboratories using in vitro a n d in vivo systems 26. In view of the fact that TGF-c~ is the major ligand, a n d T G F - a is m o r e p o t e n t t h a n E G F in certain biological responses (reviewed in ref. 12), it is essential that T G F - a also is used as ligand for studies involving E G F in b r a i n or o t h e r tissue. R e c e n t studies 9"Ls indicate both qualitative a n d q u a n t i tative differences in the kinetics of T G F - a b i n d i n g a n d E G F b i n d i n g to the E G F receptor, a n d it is possible that the p o s t - r e c e p t o r responses observed with E G F a n d with T G F - a are not identical. Moreover, the cross-reactivities of E G F a n d T G F - a a n t i s e r a should be assessed prior to their a p p l i c a t i o n to studies of p e p t i d e distribution. Finally, it seems p r u d e n t to confirm studies of E G F a n d T G F - a d i s t r i b u t i o n s by examining the a b u n d a n c e of m R N A transcripts for these related growth factors.

Acknowledgments. We wish to extend our thanks to Dr. E. Salido for

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